The phosphorylation of proteins on tyrosine residues is a posttranslational modification (PTM) with great importance for the regulation of cell proliferation, cell migration and cell differentiation. Defects in the regulation of these processes frequently caused by mutation or overexpression of protein-tyrosine kinases and associated with hyperphosphorylations participate in pathological alterations, notably in the context of malignant cell transformation. Inhibitors for constitutively active protein-tyrosine kinases have emerged as a novel class of drugs for cancer therapy over the last two decades.
Protein-tyrosine phosphatases (PTP) counteract the actions of tyrosine kinases in all pathways controlled by tyrosine phosphorylation. Therefore, the dysregulation of PTP can potentially also contribute to malignant cell transformation. Underlying mechanisms are mutations in the encoding genes which lead to altered activity, changes in PTP gene expression or alterations in mechanisms which control PTP activity. Among the latter, the reversible oxidation of PTP has found increasing attention over the last years (Östman et al., J.Biochem. 150, 345-56, 2011). The hydrolysis of phosphotyrosine by most PTP depends on an active cysteine in the PTP catalytic site. This cysteine is vulnerable to oxidation by reactive oxygen species (ROS), leading to (in principle) reversible PTP inactivation, exemplified for PTP Dep-1 (Godfrey et al., JBC, 2012; Jayavelu et al., Leukemia 2016).
A receptor tyrosine kinase (RTK) designated FLT3 is an important regulator of hematopoietic differentiation. Mutated versions of this RTK (i.e. FLT3 ITD), endowed with constitutive activity, are frequently found in patients with Acute Myeloid Leukaemia (AML). The biogenesis and localization of mutant FLT3 in AML cells is altered, a process, which contributes to transformation (Schmidt-Arras et al., Mol. Cell Biol. 25, 3690-703, 2005; Blood 113, 3568-76, 2009; Kellner et al., J Cell Mol Med 2020). Its modification could potentially present a novel strategy for interference with leukaemic cell proliferation and survival (recently reviewed in Schmidt-Arras & Böhmer, TIMM 2020; Müller & Schmidt-Arras, Cancers 2020). Research at our institute aims at a better understanding of the role of aberrantly altered signalling of PTPs and PTKs in oncogenic cell transformation.
Counteracting role of receptor PTP (RPTP) Dep-1 (PTPRJ) and CD45 (PTPRC) on wild type and mutant FLT3 receptor has been analysed in vitro (Arora et al., JBC 2011) and in vivo (Kresinsky et al., Haematologica 2018; Kresinsky et al., Oncogene 2019). Inactivation of Ptprc in a FLT3 ITD background revealed severe abnormalities of bone architecture, cell biology, and remodelling. These data indicate a previously unrecognized role of FLT3 in regulation of bone metabolism, which is affected by Ptprc.
The histone deacetylase SIRT7 was recently identified as factor regulating hematopoietic stem cell (HSC) quiescence and aging. Molecular alterations within the hematopoietic system influence longevity and differentiation of the HSC. While in juvenile HSC high levels of SIRT7 suppress mitochondrial biogenesis and respiration in HSC, in aged HSC SIRT7 expression is reduced, consequently leading to increased HSC proliferation and myeloid differentiation. Based on the observation that oncogenic FLT3 ITD suppressed SIRT7 expression, we could establish SIRT7 as an important factor in the development of age-dependent myeloid stem-cell disorders, and therefore changes of SIRT7 expression as a relevant pathomechanism in FLT3 ITD-positive AML cells (Kaiser et al., Leukemia 2020).
Deutsche Krebshilfe - Frank-D. Böhmer
Interrupting the formation of reactive oxygen species as a therapeutic approach for the FLT3-ITD positive Acute Myeloid Leukemia.
The FLT3-ITD positive Acute Myeloid Leukemia has an unfavorable prognosis and novel therapeutic options are urgently required. Treatment with FLT3-ITD selective tyrosine kinase inhibitors (TKI) has recently emerged as a suitable therapeutic modality, however, 30-40% of patients still undergo relapse even after TKI therapy or allogeneic stem cell transplantation. Previous work of the applicants of this project has identified reactive oxygen species (ROS) as contributing to leukemic cell transformation by FLT3-ITD. Elevated ROS production is at least partially mediated by activation of signal transducer of transcription 5 (STAT5) and elevated expression of NADPH oxidase 4 (NOX4), which is a direct transcriptional target of STAT5. A consequence of ROS formation is the inactivation of protein-tyrosine phosphatases (PTP), among them DEP-1/PTPRJ, a negative regulator of FLT3-ITD signal transduction. NOX4 inhibition by genetic means or using NOX4-inhibiting compounds interrupts this pathway, restores PTP activity, and markedly attenuates proliferation of FLT3-ITD positive cells in vitro and the development of a leukemia-like disease in mouse models (Jayavelu et al., NOX4-driven ROS formation mediates PTP inactivation and cell transformation in FLT3ITD-positive AML cells. Leukemia. 2016, 30(2):473-83. Jayavelu et al., NOX-driven ROS formation in cell transformation of FLT3-ITD-positive AML. Exp Hematol. 2016, 44(12):1113-1122.).
Aim of the project is assessing the possibility of interfering with ROS formation as a novel therapeutic concept in FLT3-ITD positive AML.
1) The critical role of NOX-dependent ROS formation for generation and maintenance of FLT3-ITD positive AML shall be further validated.
2) The interruption of NOX-driven ROS formation shall be tested for efficiency in combination with the application of cytostatic agents and FLT3-ITD selective TKIs.
Involved people: Anne Kresinsky, Frank-D. Böhmer in cooperation with Muhammed Burak Demircan, Florian Heidel (Abteilung Hämatologie und Internistische Onkologie des UKJ)
Grant Support: Deutsche Krebshilfe, 2017 - 2020
DFG - Jörg Müller
In vivo role of FLT3-CD45 signalling for bone remodelling
The aim of the previous project was to assess the role of the two transmembrane, receptor-like protein tyrosine phosphatases PTPRJ/DEP-1 and PTPRC/CD45 in regulating transformation of myeloid cells by the acute myeloid leukaemia (AML)-related oncoprotein FLT3-ITD in vivo. Using a disease-specific mouse model (FLT3-ITD), we found that loss of either of these PTPs aggravated a myeloproliferative disease, supporting their tumour-suppressor role. These studies have been published in Hematologica and Oncogene. In addition to aberrant haematopoiesis, the inactivation of the Ptprc gene in FLT3-ITD mice resulted in a marked and unexpected bone phenotype. Comprehensive analysis revealed that a similar, but milder phenotype was already present in the FLT3-ITD knock-in background. Our pilot studies revealed severe abnormalities of bone architecture, cell biology, and remodelling in the latter mice, which were even more pronounced with inactivation of Ptprc. The significant alteration of parameters of bone formation or resorption indicated potentially altered osteoblast (OB) and osteoclast (OC) function. These data indicated a previously unrecognized role of FLT3 in regulation of bone metabolism, which is affected by Ptprc.
The aim of this proposal is to unravel the mechanisms underlying the FLT3/ FLT3-ITD-Ptprc-mediated signalling axis to control bone formation and remodelling. Molecular analysis of the relevant bone cell types, their stem cell biology and repopulation capacity and the involved signalling mechanisms are the focus of our studies to understand how FLT3 and Ptprc regulate bone metabolism. Our previously established FLT3-ITD/ Ptprc-/-mouse strain and the corresponding WT, FLT3-ITD and Ptprc-/- control strains represent the technical basis for this proposal.
We will address the following objectives:
Characterization of the bone phenotype of FLT3-ITD expressing mice in detail,
Determination of the role of FLT3-ITD and CD45 in bone cell differentiation and function,
Analysis of the underlying pathways of the altered bone cell function in FLT3-ITD and FLT3-ITD Ptprc-/- mice and
Elucidation of the origin of ectopic bone formation and aberrant HSC homing.
Taken together, detailed characterization of the phenotype of the FLT3-ITD Ptprc-/- mice in comparison to wild-type mice and mice carrying single mutations provides mechanistic insights, how FLT3 and Ptprc control bone remodelling, the HSC niche, and mutual interactions of both. Detailed understanding of these processes for normal and pathological haematopoiesis and bone remodelling offers the potential for targeted intervention for bone and hematologic disorders and may translate into improved outcome for affected patients.
Cooperation: The project will be realized in close collaboration with Prof. Lorenz C. Hofbauer, Universitätsklinikum Dresden der Technischen Universität Dresden, Bereich Endokrinologie, Diabetes & Osteologie and Prof. Martina Rauner, Group Leader and Scientific Direktor of the „Bone Lab“, Universitätsklinikum Carl Gustav Carus, TU Dresden. Local close collaboration with the Department of Haematology and Oncology of the Jena University Hospital.
Funding: DFG Mu955/14-1, 2020-2023
DFG - Jörg Müller
Role of SIRT7 in FLT3 ITD driven cell differentiation and transformation
Oncogenic ITD mutations in the receptor tyrosine kinase FLT3 represent one of the important classes of driver mutations in Acute Myeloid Leukemia (AML) patients. Despite approval of Midostaurin as inhibitor of this oncogene, FLT3 ITD positive AML patients are still associated with poor prognosis. The function of FLT3 in promoting myeloid development remains poorly defined, despite being commonly mutated in AML. Defining differentiation pathways is crucial to understand pathogenesis of this hematopoietic disorder. Our previous work indicates for the first time a molecular correlation of FLT3 ITD-based development of myeloid neoplasms to the expression of SIRT7 as factor controlling HSC quiescence.
The foundation of this project application are our novel findings about the role of SIRT7 in myeloid stem-cell disorders, which were (recently published) in Leukemia (Kaiser et al., 2020). Our work established SIRT7 as important factor in the development of age-dependent myeloid stem-cell disorders and therefore changes of SIRT7 expression as a relevant pathomechanism in FLT3 ITD-positive AML cells. AML patients with low SIRT7 expression have poor prognosis demonstrating the clinical relevance of this protein for patient survival. SIRT7 was recently identified as a factor regulating hematopoietic stem cell (HSC) quiescence and aging. Molecular alterations within the hematopoietic system influence cellular longevity and differentiation.
The aim of this proposed project is to dissect the mechanism, how FLT3 ITD mutations induce cell differentiation by affecting SIRT7 expression and thereby driving accumulation of myeloid expansion of blast cells in vivo. By using primary AML patient samples, established in vitro model cell systems and mouse models we will study the role of SIRT7 on FLT3 ITD-mediated myeloid aberrancies and clonal expansion. After establishing molecular correlation of FLT3 ITD-SIRT7-signalling on HSC differentiation/ transformation rejuvenation strategies of HSC (i.e. SIRT7 activation) will be envisaged. This could make it possible to control FLT3 ITD-mediated imbalances of HSC quiescence and subsequently suppression of myeloid differentiation FLT3 ITD AML patients in the future.
SIRT7 is reduced in AML (FLT3 ITD) cells (cell lines, primary samples, mouse BM cells).
Suppression of SIRT7 is FLT3 ITD kinase activity dependent.
C/EBP-mediated control of SIRT7 expression is negatively regulated by FLT3 ITD.
Restoration of SIRT7 induces cell differentiation and suppresses cell transformation.
Characterize the role of FLT3 ITD on SIRT7 activity
Analyse the mechanism, how SIRT7 mediates FLT3 ITD-driven cell transformation/ cell differentiation on cellular level
Assess function of SIRT7 on FLT3 ITD-driven cell transformation/ hematopoietic differentiation in vivo
Affecting FLT3 ITD mediated cell transformation by activation of the cellular protein acetylation
Cooperations: Close collaboration with the Department of Haematology and Oncology of the Jena University Hospital.