INTEGRATIVE FUNCTIONAL ANALYSIS OF THE DEK-NUP214 FUSION PROTEIN IN ACUTE MYELOID LEUKAEMIA
Author(s): ,
Fabio Liberante
Affiliations:
Institute for Medical Biochemistry,University of Veterinary Medicine,Vienna,Austria;Ludwig Boltzmann Institute for Cancer Research,Vienna,Austria
,
Konstantina Chatziathanasiou
Affiliations:
Institute for Medical Biochemistry,University of Veterinary Medicine,Vienna,Austria;Ludwig Boltzmann Institute for Cancer Research,Vienna,Austria
Florian Grebien
Affiliations:
Institute for Medical Biochemistry,University of Veterinary Medicine,Vienna,Austria;Ludwig Boltzmann Institute for Cancer Research,Vienna,Austria
EHA Library. Liberante F. Jun 14, 2019; 265999; PF209
Dr. Fabio Liberante
Dr. Fabio Liberante
Contributions
Abstract

Abstract: PF209

Type: Poster Presentation

Presentation during EHA24: On Friday, June 14, 2019 from 17:30 - 19:00

Location: Poster area

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.

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.

Session topic: 3. Acute myeloid leukemia - Biology & Translational Research

Keyword(s): AML, Fusion, Leukemogenesis, Proteomics

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