Identifikation kritischer Faktoren für die biomolekulare Kondensation in NUP98::KDM5A-getriebener akuter myeloischer Leukämie mittels FRET-basierter Methode

The complexity of AML is reflected by the high variety of genes involved in disease development and propagation, and this complicates the development of targeted therapy. The NUP98 gene, which encodes for the Nuclear Pore Complex (NPC) protein Nucleoporin 98, is involved in chromosomal rearrangements in roughly 4% of pediatric AML. The fusion of the N-terminus of NUP98 to one of its 31 distinct partner genes, such as KDM5A, prevents myeloblasts from undergoing normal differentiation into mature blood cells and the resulting NUP98::KDM5A fusion oncoprotein drives leukemia development. Recently, liquid-liquid phase separation (LLPS), has been proposed to represent a disease-causing pathomechanism. This process drives the formation of NUP98::KDM5A-containing biomolecular condensates upon self-solvation of condensate-prone biomolecules within a homogeneous solution. Despite emerging interest in studying biomolecular condensates in NUP98-fusion-driven pediatric AML, it is unclear what macromolecules drive the formation of NUP98::KDM5A-contining onco-condensates. We aim to identify factors within the condensate-specific NUP98::KDM5A-interactome that are critical for the formation and oncogenic functions of NUP98::KDM5A-driven biomolecular condensates. We established a confocal microscopy-based method relying on the close interactions of proteins within biomolecular condensates taking advantage of Förster resonance energy transfer (FRET) to evaluate the proximity and interaction strength of NUP98::KDM5A within biomolecular condensates. To study FRET, we created constructs enabling the expression of a NUP98::KDM5A fusion protein coupled to fluorophores with a spectral overlap. This allows energy to be transferred from the donor to the acceptor upon close proximity. In preliminary experiments, we were able to show efficient FRET and thus proof of the concept proposed by us.To use the FRET-based readout for biomolecular condensation of NUP98::KDM5A at higher throughput, we will couple FRET to flow cytometry (Flow-FRET). The combination of Flow-FRET with an arrayed CRISPR/Cas9 screening approach will be used to identify factors that are essential for biomolecular condensation in NUP98::KDM5A expressing cells. Preliminary experiments performed in our laboratory using enzyme-catalyzed proximity labeling by a biotin ligase (bioID) lead to the identification of proteins colocalizing with NUP98::KDM5A within biomolecular condensates. We will select candidates from the NUP98::KDM5A bioID dataset and investigate their contribution to NUP98::KDM5A condensate formation using a CRISPR/Cas9-mediated knockout screen using the Flow-FRET read-out. The top 3-5 candidates from this screen will be subjected to further in-depth mechanistic analyses in cell lines and animal models of NUP98::KDM5A driven AML. We will investigate the functional consequences of loss of selected high-confidence candidates on AML cell physiology by evaluating changes in cell proliferation, survival and differentiation. Further, visualization of dynamic changes in the subcellular localization patterns of NUP98::KDM5A condensates upon loss of candidate condensate-residing proteins using confocal microscopy will be performed. Together, this project will lead to novel insights and potentially new therapeutic options to target proteins that are essential for onco-condensate formation in NUP98::KDM5A driven pediatric AML.

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VetCore

Childrens Cancer Research Institute (CCRI)
Funktion: Partner
Abteilung: FlowCore