PF209 INTEGRATIVE FUNCTIONAL ANALYSIS OF THE DEK‐NUP214 FUSION PROTEIN IN ACUTE MYELOID LEUKAEMIA

Abstract

Background:Acute myeloid leukemia (AML) is often driven by oncogenic fusion proteins that result from chromosomal rearrangements. A better functional understanding of AML fusion proteins is critical to improving treatment, as patients usually present with a very poor prognosis. The t(6;9) rearrangement gives rise to the DEK‐NUP214 fusion protein, which drives malignant transformation. The mechanistic basis of DEK‐NUP214‐induced leukemia is unclear and currently, there are no therapies specifically targeting it.Aims:We aim to investigate molecular mechanisms of DEK‐NUP214‐induced leukemogenesis via integrative analysis of its proteomics‐based interactome. DEK‐NUP214‐interacting proteins will be functionally investigated via targeted CRISPR/Cas9‐knockout screening in human cells. Candidate molecular mechanisms will be validated in in vivo models of DEK‐NUP214 leukemia.Methods:We used CRISPR/Cas9‐mediated genome engineering to introduce epitope tags into the endogenous DEK‐ and DEK/NUP214 genes in leukemia cell lines. DEK‐ and DEK‐NUP214 protein complexes were isolated from cells via affinity purification after careful optimization of biochemical protocols. Protein complexes were analyzed by mass spectrometry (AP‐MS). The human DEK‐NUP214‐expressing cell line FKH‐1 was engineered to stably express functional Cas9. Retroviral expression of DEK‐NUP214 in mouse hematopoietic progenitor cells was used to establish in vivo AML models of t(6;9) AML.Results:Using Cas9/crRNA ribonucleoprotein complexes, we have successfully edited, isolated and validated leukemia cell lines to harbor endogenous Strep‐HA‐tagged DEK alleles. AP‐MS analysis of the DEK protein interactome revealed 127 unique, high‐confidence interaction partners. In line with its proposed function the DEK interactome is highly enriched in RNA‐binding‐ and nucleolar proteins. In addition, we find that DEK interacts with protein complexes involved in epigenetic regulation. AP‐MS studies of the leukemogenic fusion DEK‐NUP214 identified 228 high‐confidence unique interactors, including members of the condensin (SMC2/4/NCAPH) and eIF3 complexes, both previously unpublished interactors.46 proteins interacted with both DEK and DEK‐NUP214 with high significance. The resulting protein set is enriched in members of the Nop56p‐associated pre‐rRNA complex, as well as the catalytic subunit of DNA‐dependent Protein Kinase (PRKDC) and the casein kinase II complex. This suggests roles for DEK‐NUP214 in DNA damage and genome instability, and may afford a new clinical opportunity, as there are a number of DNA‐PK inhibitors already in clinical trials for other cancer types. Consistent with the interaction with casein kinase II, phospho‐analysis of AP‐MS datasets identified that serine 32 of DEK and DEK‐NUP214 is phosphorylated. This phosphorylation site can trigger DEK multimerization, suggesting that DEK‐NUP214 may also be present in a multimeric state.We are currently validating potential mechanistic contributions of the identified proteins as requirements for leukaemogenesis in the t(6;9) FKH‐1 cell line through CRISPR/Cas9‐ and shRNA‐mediated gene silencing. In parallel, we are developing mouse models of DEK‐NUP214‐driven AML that will be used for in vivo validation studies.Summary/Conclusion:Integrative analysis of the DEK‐ and DEK‐NUP214 interactomes has already unearthed a number of insights into the potential leukemogenic effect of this fusion oncoprotein. Furthermore, it has identified a number of targets that could provide clinically‐relevant opportunities for t(6;9) AML patients.