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Identification of actionable dependencies among direct transcriptional gene targets of the NUP98-JARID1A fusion protein in Acute Myeloid Leukemia

Abstract
Oncogenic fusion proteins arise from structural chromosomal rearrangements and are a common characteristic of acute myeloid leukemia (AML). Fusion proteins involving the Nucleoporin 98 (NUP98) gene are recurrently found in AML and are enriched in pediatric patients. NUP98-JARID1A is the most frequent NUP98-rearrangement in infant leukemia and is associated with particularly poor prognosis with no targeted therapy being available. Hence, there is an urgent need to unravel the underlying molecular mechanisms of NUP98-JARID1A leukemogenesis to empower the development of tailored treatments.To investigate functional genetic dependencies in NUP98-JARID1A-driven AML, we conducted a genome-scale CRISPR/Cas9 loss-of-function screen using a NUP98-JARID1A-dependent AML cell line. This analysis identified 2550 genes that are required for the proliferation and survival of NUP98-JARID1A-dependent AML. As we and others have shown that NUP98-JARID1A can act as a direct regulator of gene expression, we propose that specific effectors of the oncogenic program of NUP98-JARID1A are under direct transcriptional control of the fusion protein. Therefore, functional annotation of direct target genes of NUP98-JARID1A for critical effectors of its leukemogenicity will identify novel mechanistic insights into NUP98-fusion-driven AML that will improve management strategies of patients suffering from this disease.Therefore, we are establishing a new model for ligand-mediated degradation of NUP98-JARID1A utilizing the degradation tag (dTAG) system. The induced recruitment of the CRBN E3 ubiquitin ligase complex followed by proteasomal degradation will allow fast and efficient depletion of the fusion protein and the subsequent investigation of immediate changes in cellular processes. Using this model we will perform SLAM-seq to capture early and subtle changes in transcriptional changes by measurement of newly synthesized mRNA upon NUP98-JARID1A degradation. Genes under direct transcriptional control of the oncogenic fusion protein will be rapidly downregulated after NUP98-JARID1A degradation and can thereby be identified as direct transcriptional targets. I will intersect the list of genetic dependencies in NUP98-JARID1A-driven leukemia obtained by the genome-wide CRISPR/Cas9 screen with the genes whose expression is directly regulated by NUP98-JARID1A. This will enable the identification of functional genetic dependencies in NUP98-JARID1A-driven AML that are under direct transcriptional control of the oncogenic fusion protein.Confirmed target genes will be ranked according to different criteria, including novelty, druggability and potential mechanism of action. Selected high-confidence candidates will be further investigated in in-depth mechanistic analyses using genetic (e.g. CRISPR/Cas9-mediated knock out) as well as pharmacologic approaches (eg. small molecule inhibitors) in a variety of cell lines, PDX mouse models and in cells from primary AML patient samples. This project will provide new insights into the molecular mechanisms of NUP98-JARID1A-driven leukemia and will identify actionable targets that could improve treatment of patients suffering from this disease.
Kurzbezeichnung
Identification of NUP98-JARID1A targets
Projektleitung
Tröster Selina
Laufzeit
01.12.20-30.11.23
Programm
ÖAW DOC
Art der Forschung
Grundlagenforschung
Beteiligte Vetmed-Organisationseinheiten
Institut für Medizinische Biochemie
Gefördert durch
Österreichische Akademie der Wissenschaften, Dr. Ignaz Seipel-Platz 2, 1010 Wien, Österreich
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