Targeting Kindlin-3-mediated integrin activation and signaling to eradicate persistent MPN clones

• Applicant: Professor Dr. Steffen Koschmieder
• Subject Area: Hematology, Oncology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 517204983

In Myeloproliferative neoplasms (MPN), constitutive activation of the JAK-STAT and other oncogenic pathways in malignant hematopoietic stem cells (HSC), their resistance to JAK inhibitors such as Ruxolitinib (Rux), and remodeling of the microenvironment (ME) and progression to myelofibrosis and acute leukemia significantly decrease survival rates of MPN patients. Targeting the interaction between malignant HSC and their ME, which is controlled by integrin-mediated adhesion, may offer new and more effective MPN treatments. We have recently demonstrated the essential role of Kindlin-3 (K3), a crucial regulator of integrin activity, in chronic myeloid leukemia (CML). Using BM chimeras, we demonstrated that K3 is crucial for integrin activity, BM retention, and survival of CML HSC. K3 depletion significantly reduced leukemic burden and prolonged survival in transgenic CML mice, and the combination of K3 depletion and Bcr-Abl (BA) tyrosine kinase inhibition further improved treatment outcomes. In further preliminary experiments, we found STAT3- and AKT-induced overexpression of K3 in BA-negative MPN patient samples and JAK2V617F-overexpressing HSC lines. In the current project, we will analyze the functional role of K3 and its integrin-associated partners in the pathogenesis and progression of Bcr-Abl negative MPN and during Rux failure, by utilizing unique K3-proficient and -deficient MPN mouse models as well as primary cells and CRISPR-Cas9 engineered K3-depleted induced pluripotent stem cells (iPSC) derived from MPN patients. Cells will be analyzed either in vivo or in in vitro in iPSC-coculture systems by spatial transcriptomics using the GeoMx Digital Spatial Profiler system and by functional assays in order to elucidate the connections between K3-mediated integrin adhesion of MPN and ME cells. The project pursues the following objectives: Aim 1A will define how JAK2V617F-HSC interact with their ME before and during Rux treatment, using irradiation-free BM chimeras and spatial gene expression analysis to study integrin-ligand interactions in vivo, and in vitro adhesion, co-culture, and survival assays using murine MPN HSC. Aim 1B will explore the impact of K3 depletion on disease progression and Rux treatment response in MPN mice. Aims 1C and 2A will analyze the transcriptional regulation of K3 to identify novel therapeutic strategies to antagonize K3 overexpression in MPN. This will involve K3 reporter constructs, CRISPR-Cas9-mediated gene targeting, and specific drugs to inhibit critical signaling pathways and transcription factors in oncogene-transformed HSC cell lines and iPSCs. Additionally, we will investigate K3 expression in MPN patient blood and BM and correlate it with clinical parameters. Aim 2B will assess the functional role of K3 in human MPN cells, using K3-deficient iPSC cells in 3D cocultures and analyzing the effect of K3 depletion on MPN cell adhesion, survival, and differentiation.

• DFG Programme: Research Units
• Subproject of: FOR 5659: TARGET-MPN: TARgetinG disease pErsisTence and progression of MyeloProliferative Neoplasms (MPN)
• International Connection: Austria
• Partner Organisation: Fonds zur Förderung der wissenschaftlichen Forschung (FWF)
• Cooperation Partner: Dr. Peter Krenn