The main focus of our research is to understand the mechanisms responsible for leukemia maintenance, progression and therapeutic response. We are mainly interested in hematological malignancies such as CML, AML or MPNs which are primarily due to activation of oncogenic pathways. Using cell culture and mouse models based experimental approaches we aim to identify the oncogene addicted signaling pathways to develop precise targeted therapies in leukemias. In addition we have a close collaboration with the UCCSH molecular tumor board (MTB ) which performs the genetic and molecular analysis from patients’ samples to determine novel variants responsible for cancer progression. We are interested in the functional characterization of novel genetic variants in different malignancies. Furthermore, we are also focused on understanding mechanisms of drug resistance. We aim towards the development of alternative therapeutic approaches in relapsed disease settings. Examples of our ongoing projects are given below:
Project 1: The significance of the IL-3 receptor beta chain for FTL3-ITD dependent oncogenic signalling in AML
In acute myelogenous leukemia (AML), activating mutations of the receptor tyrosine kinase FLT3 can be found in about one third of patients (Birg et al., 1992). Internal tandem domain duplications (ITD) are most common, the remainder are mutations of the tyrosine kinase domain (TKD) (Thiede et al., 2002). FLT3-ITD is associated with a dismal prognosis – only 20 per cent of patients survive for 4 years (Schlenk et al., 2008). The FLT3 tyrosine kinase inhibitor midostaurin improved survival in FLT3 mutated AML when added to standard chemotherapy (Stone et al., 2017), and was recently approved for treatment of adult patients with newly diagnosed FLT3 mutated AML. We have previously shown that resistance to FLT3 kinase inhibitors can be mediated by secondary FLT3 mutations obviating drug binding to its target in vitro (von Bubnoff et al. 2009). However, we noted that the majority of cell lines with acquired resistance to one of the FLT3 inhibitors midostaurin or sorafenib did not harbor secondary resistance mutations in FLT3 (von Bubnoff et al. 2009). Also, only a proportion of patients with FLT3 inhibitor resistant AML displays secondary FLT3 mutations (Heidel et al., 2006; Smith et al., 2017; von Bubnoff et al., 2010; Zhang et al., 2019). In this project, we aim to understand the FLT3 kinase-independent resistance to FLT3 inhibitors in order to explore strategies to overcome clinical resistance to FLT3 inhibitors in FLT3 mutated AML (Rummelt et al. 2020). With this goal, we have found that FLT3-ITD directly interacts and phosphorylates CSF2RB (colony stimulating factor 2 beta chain) and that downregulation of CSF2RB leads to the attenuation of FLT3-ITD mediated AML in mouse models (Charlet et al. 2021). Based on these results, we proposed a model where FLT3-ITD phosphorylates the CSF2RB which constitutively associated with JAK family kinases and contribute to STAT5 activation (Figure 1). Our ongoing work further defines the clinical significance of CSF2RB in FLT3 mediated AML disease pathogenesis and relapse towards the FLT3 kinase inhibitors in collaboration with Prof. Dr. Gabriela Riemekasten from the Department of Rheumatology and Clinical Immunology, UKSH, Campus Lübeck for MicroScale Thermophoresis (MST) binding assay. We have collaborate with Prof. Dr. Timo Gemoll, University of Lübeck, Sektion für Translationale Chirurgische Onkologie & Biomaterialbanken, Klinik für Chirurgie for Mass spectrometry using Electrospray Ionization (ESI-MS) in order to identify the interactome between FLT3-ITD and CSF2RB.
Project 2: Role of ER stress in CSF3R mutations mediated chronic neutrophilic leukemia (CNL)
Chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) are rare hematological malignancies that exhibit typical clinicopathologic characteristics such as expansion of neutrophils in the bone marrow and peripheral blood as well as splenomegaly. Recent advancement in sequencing technologies have led to the identification of oncogenic mutations in the granulocyte-colony stimulating factor 3 receptor (CSF3R) in CNL and aCML patients (approximately 50%) (Pardanani et al. 2015). In addition to CNL, CSF3R mutations are also found in patients with severe congenital neutropenia (SCN) at lower frequencies and in 1% of de novo AML patients (Maxon J et al. 2013). Identification of CSF3R mutations in these malignancies has put an emphasis on understanding the disease mechanism and of kinases, which play a major role in disease progression are crucial for an efficient targeted therapy (Figure 2). Our ongoing work is to delineate the role of oncogenic specific mechanism of CSF3R mutations mediated CNL using patients’ samples and mouse models. In this project, we have collaboration with Prof. Hauke Busch University of Lübeck, LIED, Lübecker Institut für Experimentelle Dermatologie, Department of systems biology for RNA-seq analysis in order to identify the crucial signal pathways upregulation from CNL patient samples. In addition, we have also collaboration with MPN-GSG (Aachen) for acquiring MPN patient samples.
Project 3: Understanding the ruxolitinib (JAKAVI) resistance mechanisms and identification of novel therapeutic approaches in JAK2V617F mediated myeloproliferative neoplasms (MPNs)
JAK2 is a cytoplasmic tyrosine kinase, which plays a major role in hematopoiesis and cytokine mediated signaling (Parganas et al., 1998) (Neubauer et al., 1998). The occurrence of the somatic activation mutation valine to phenylalanine in the pseudokinase domain (V617F) of JAK2 has been implicated in myeloproliferative neoplasms including polycythemia vera (PV) (PV; 90%), essential thrombocythemia (ET; 50%) and primary myelofibrosis (PMF; 50%) (James et al., 2005) (Baxter et al., 2005; Kralovics et al., 2005; Levine et al., 2005). In addition to MPNs, the JAK2V617F mutation has also been observed at very low frequencies in myelodysplastic syndrome, chronic myelomonocytic leukemia (3-8%) and very rarely in systemic mastocytosis (Levine et al., 2005a; Steensma et al., 2005). A subset of PV patients, negative to V617F mutation, showed a gain of function mutations affecting the exon 12 of JAK2 (Scott et al., 2007). Alteration of these residues in JAK2 leads to constitutive activation of JAK2 and STAT5 signaling pathways. These discoveries encouraged the development of small molecular inhibitors against the JAK2. Several JAK2 inhibitors including ruxolitinib, fedratinib and lestaurtinib displayed remarkable activity against the JAK2 mediated MPNs both in vitro and in mouse models (Pardanani et al., 2007; Santos et al., 2010; Verstovsek et al., 2010). Because of this success, the FDA has approved ruxolitinib for the treatment of PMF and PV patients. However, development of resistance against the targeted therapies is inevitable and previously it has been well demonstrated in case of CML, AML and GIST. With this observation, we aim to develop ruxolitinib resistant clones to understand the possible mechanism of resistance and also aim to develop novel therapeutic agents against the ruxolitinib resistant MPNs. To this end, we have identified a novel JAK2 variant with a molecular weight of 45kDa (Figure 3). We focused on understanding the mechanism of this 45-kDa JAK2 variant activation and drug resistance phenotype. In association with molecular tumor board (MTB), we aim to identify possible novel JAK2 variants in MPN patients.
Project 4: Understanding the role of p-loop conformational states in FIP1L1-PDGFRA kinases which regulates the phosphatases interaction
The idiopathic hyper eosinophilic syndrome (HES) and chronic eosinophilic leukemia (CEL) are defined by persistent hypereosinophilia. The generation of constitutively activated fusion kinase FIP1L1-PDGFRA due to chromosomal translocation is one of the oncogenic drivers in CEL (Cools et al., 2003). Approximately, 10 to 20 % of CEL patients are positive for FIPL1L-PDGFRA (Gotlib and Cools, 2008). Imatinib is clinically effective in FIP1L1-PDGFRA positive patients, however, imatinib treatment leads to the development of drug resistance in CEL patients (Cools et al., 2003) (von Bubnoff et al., 2005) (Ohnishi et al., 2006) (Gotlib and Cools, 2008) (Salemi et al., 2009) (Lierman et al., 2012). In our previous work, we have established a comprehensive cell-based screening strategy to identify potential drug resistance mechanisms against PDGFRA inhibitors such as imatinib, nilotinib and sorafenib in a FIP1L1-PDGFRA mediated CEL model (von Bubnoff et al., 2011). Interestingly, we have detected F604S (phe to ser at 604) to be the most abundant mutation in our cell-based screening method against imatinib (Gorantla et al., 2015) (Figure 4). Using biochemical methods, we could demonstrate that F604S mutation leads to decreased autophosphorylation of FIP1L1-PDGFRA by dephosphorylation mediated by phosphatases and stabilization of the FIP1L1-PDGFRA protein. However, F604S exchange selectively activates STAT5 similar to wild type even though it is less auto phosphorylated. Interestingly, imatinib resistant CEL patients who harbor L629P also showed similar protein stabilization by decreasing the target kinase autophosphorylation (Gorantla et al., 2015). Based on these observations, we could conclude that the stabilization of FIP1L1-PDGFRA protein is a frequent drug resistance mechanism in CEL. In this project, we aim to elucidate the structural and molecular mechanisms by which F604S/L629P exchange confers the protein stabilization by performing molecular dynamic simulations and cryo-EM in order to gain in depth understanding of how p-loop mutations alter the signaling pathways. We have close a collaboration with Prof. Jeroen Mesters, University of Lübeck, Department of Biochemistry for protein modeling and structural analysis of PDGFRA.
Project 5: Characterization of tumor associated markers in extracellular vesicles derived from leukemia models
Extracellular vesicles (EVs) are tiny membrane bound particles which are well known for their pivotal role in cell-cell communication especially in disease conditions. EVs are secreted by every eukaryotic cell type and based on their size and origin, they are mainly categorized into Exosomes (50-100 nm), Ectosomes (100-800 nm) and apoptotic bodies (500-4000 nm). EVs acts as a short-range intercellular communication or long-range communication when released into the bloodstream through which they carry a cargo molecule such as DNA, RNA or proteins, lipids, and metabolites of the origin cells. The presence of the EVs in biological fluids makes them an excellent candidate as a diagnostic or prognostic marker in several diseases especially in tumors via noninvasive or minimal invasive procedures. Among different types of EVs, ectosomes mimic the tumor cell surface as they are released directly from tumor cells by membrane blebbing. To decipher the clinical value of leukemia-derived EVs as a prognostic or predictive biomarkers, we aim to identify tumor associated markers in different leukemia models (Figure 5). With the help of Prof. Dr. Frank Gieseler in our department who has vast experience in the EV field and employing methods such as sequential centrifugation, Flow cytometry and Nanoparticle Tracking Analysis (NTA), we aim to determine leukemia specific biomarkers from ectosome like EVs.