Dataset Information

SMARCA5 interacts with NUP98-NSD1 oncofusion protein and sustains hematopoietic cells transformation.

ABSTRACT:

Background

Acute myeloid leukemia (AML) is characterized by accumulation of aberrantly differentiated hematopoietic myeloid progenitor cells. The karyotyping-silent NUP98-NSD1 fusion is a molecular hallmark of pediatric AML and is associated with the activating FLT3-ITD mutation in > 70% of the cases. NUP98-NSD1 fusion protein promotes myeloid progenitor self-renewal in mice via unknown molecular mechanism requiring both the NUP98 and the NSD1 moieties.

Methods

We used affinity purification coupled to label-free mass spectrometry (AP-MS) to examine the effect of NUP98-NSD1 structural domain deletions on nuclear interactome binding. We determined their functional relevance in NUP98-NSD1 immortalized primary murine hematopoietic stem and progenitor cells (HSPC) by inducible knockdown, pharmacological targeting, methylcellulose assay, RT-qPCR analysis and/or proximity ligation assays (PLA). Fluorescence recovery after photobleaching and b-isoxazole assay were performed to examine the phase transition capacity of NUP98-NSD1 in vitro and in vivo.

Results

We show that NUP98-NSD1 core interactome binding is largely dependent on the NUP98 phenylalanine-glycine (FG) repeat domains which mediate formation of liquid-like phase-separated NUP98-NSD1 nuclear condensates. We identified condensate constituents including imitation switch (ISWI) family member SMARCA5 and BPTF (bromodomain PHD finger transcription factor), both members of the nucleosome remodeling factor complex (NURF). We validated the interaction with SMARCA5 in NUP98-NSD1⁺ patient cells and demonstrated its functional role in NUP98-NSD1/FLT3-ITD immortalized primary murine hematopoietic cells by genetic and pharmacological targeting. Notably, SMARCA5 inhibition did not affect NUP98-NSD1 condensates suggesting that functional activity rather than condensate formation per se is crucial to maintain the transformed phenotype.

Conclusions

NUP98-NSD1 interacts and colocalizes on the genome with SMARCA5 which is an essential mediator of the NUP98-NSD1 transformation in hematopoietic cells. Formation of NUP98-NSD1 phase-separated nuclear condensates is not sufficient for the maintenance of transformed phenotype, which suggests that selective targeting of condensate constituents might represent a new therapeutic strategy for NUP98-NSD1 driven AML.

SUBMITTER: Jevtic Z

PROVIDER: S-EPMC8785526 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nuclear receptor-binding SET domain protein 1 (NSD1) prototype is a family of mammalian histone methyltransferases (NSD1, NSD2/MMSET/WHSC1, NSD3/WHSC1L1) that are essential in development and are mutated in human acute myeloid leukemia (AML), overgrowth syndromes, multiple myeloma and lung cancers. In AML, the recurring t(5;11)(q35;p15.5) translocation fuses NSD1 to nucleoporin-98 (NUP98). Here, we present the first characterization of the transforming properties and molecular mechanisms of NUP98-NSD1. We demonstrate that NUP98-NSD1 induces AML in vivo, sustains self-renewal of myeloid stem cells in vitro, and enforces expression of the HoxA7, HoxA9, HoxA10 and Meis1 proto-oncogenes. Mechanistically, NUP98-NSD1 binds genomic elements adjacent to HoxA7 and HoxA9, maintains histone H3 Lys 36 (H3K36) methylation and histone acetylation, and prevents EZH2-mediated transcriptional repression of the Hox-A locus during differentiation. Deletion of the NUP98 FG-repeat domain, or mutations in NSD1 that inactivate the H3K36 methyltransferase activity or that prevent binding of NUP98-NSD1 to the Hox-A locus precluded both Hox-A gene activation and myeloid progenitor immortalization. We propose that NUP98-NSD1 prevents EZH2-mediated repression of Hox-A locus genes by colocalizing H3K36 methylation and histone acetylation at regulatory DNA elements. This report is the first to link deregulated H3K36 methylation to tumorigenesis and to link NSD1 to transcriptional regulation of the Hox-A locus. Experiment Overall Design: Total RNA was extracted from stably transformed progenitors cultured in vitro and the expression levels of mRNA transcripts quantified using the Affymetrix GeneChip Mouse Genome 430 2.0 array, as previously described. The GEO database accession numbers: for progenitors immortalized by HoxA9 (GSM190542, GSM190546, GSM190547); for progenitors immortalized by coexpressed HoxA9 plus Meis1 (GSM190548, GSM190549, GSM190550); for progenitors immortalized by NUP98-NSD1 (GSM190551, GSM190552, GSM190553); and for progenitors immortalized by MLL-ENL (GSM190554). Experiment Overall Design: NOTE: CEL files and dChip data were requested by GEO but not provided.