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NUP98-HOXA10hd fusion protein sustains multi-lineage haematopoiesis of lineage-committed progenitors in transplant setting.

ABSTRACT: Objectives: Exploring approaches of extending the haematopoiesis time window of MPPs and lineage-committed progenitors might produce promising therapeutic effects. NUP98-HOXA10hd (NA) fusion protein can expand long-term haematopoietic stem cells (HSCs) and promote engraftment competitiveness without causing obvious oncogenesis. Our objectives were to investigate the roles of NA fusion protein in MPP and downstream lineage-committed progenitor context.

Material and methods: 300 sorted MPPs (Lin^- CD48^- c-kit⁺ Sca1⁺ CD135⁺ CD150^- ) were mixed with 5 × 10⁵ total BM helper/competitor cells and injected into irradiated recipients. For secondary transplantation, 5 × 10⁶ total BM cells from primary recipient mice were injected into lethally irradiated recipients. NA-MPP recipient mice were sacrified for flow cytometric analysis of bone marrow progenitors at indicated time points. Sorted MPPs and myeloid progenitors were used for RNA-seq library preparation.

Results: We showed that NA-expressing MPPs achieved significantly longer multi-lineage haematopoiesis (>44-week) than natural MPPs (20-week). NA upregulated essential genes regulating long-term haematopoiesis, cell cycle, epigenetic regulation and responses to stress in MPPs. These molecular traits are associated with the earlier appearance of a Sca1^- c-kit⁺ myeloid progenitor population, and more abundant cellularity of lineage-committed progenitor as well as bone marrow nucleated cells. Further, the NA-derived primary bone marrow cells, which lack NA-LSK cells, successfully repopulated secondary multi-lineage haematopoiesis over 20 weeks.

Conclusions: This study unveiled that NA fusion protein promotes MPP and lineage-committed progenitor engraftment via extending long-term multi-lineage haematopoiesis.

SUBMITTER: Dong Y

PROVIDER: S-EPMC7507399 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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NUP98-HOXA10hd fusion protein sustains multi-lineage haematopoiesis of lineage-committed progenitors in transplant setting.

Dong Yong Y Wang Kaitao K Weng Qitong Q Wang Tongjie T Zhou Peiqing P Liu Xiaofei X Geng Yang Y Liu Lijuan L Wu Hongling H Wang Jinyong J Du Juan J

Cell proliferation 20200729 9

<h4>Objectives</h4>Exploring approaches of extending the haematopoiesis time window of MPPs and lineage-committed progenitors might produce promising therapeutic effects. NUP98-HOXA10hd (NA) fusion protein can expand long-term haematopoietic stem cells (HSCs) and promote engraftment competitiveness without causing obvious oncogenesis. Our objectives were to investigate the roles of NA fusion protein in MPP and downstream lineage-committed progenitor context.<h4>Material and methods</h4>300 sorte ...[more]

PMID: 32725842

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Project description:BackgroundAcute 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.MethodsWe 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.ResultsWe 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.ConclusionsNUP98-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.

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NUP98-HOXA10hd fusion protein sustains multi-lineage haematopoiesis of lineage-committed progenitors in transplant setting.

Publications

NUP98-HOXA10hd fusion protein sustains multi-lineage haematopoiesis of lineage-committed progenitors in transplant setting.

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