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.

MPP and downstream lineage-committed progenitor context. Material and Methods: 300 sorted MPPs (Lin − CD48 − c-kit + Sca1 + CD135 + CD150 − ) were mixed with 5 × 10 5 total BM helper/competitor cells and injected into irradiated recipients. For secondary transplantation, 5 × 10 6 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 multilineage 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 Sca1c-kit + myeloid progenitor population, and more abundant

| INTRODUC TI ON
Allogeneic stem cell transplantation is widely used for blood disorders, such as leukaemia, myeloproliferative diseases, anaemia.
However, lack of timely available HLA matched-donor hindered the application of allogeneic transplantation in clinical applications. Cord blood banks hold the potential to serve as the cell source for transplantation, while the insufficient haematopoietic stem cell number in one unit of cold blood become the main obstacle for the application of cord blood. One solution is to expand haematopoietic stem cell in vitro prior to transplantation. Great efforts have been made to achieve haematopoietic stem cell (HSC) expansion, including the use of small molecules, such as SR1, UM171, UM729, as well as the introduction of intrinsic regulators into HSC-enriched stem cell population, HOXB4 for example. [1][2][3][4][5][6] However, the clinical efficacy of these approaches remains unknown and need further investigations. Another possible solution is to enhance self-renewal potential of progenitors or generate abundant progenitor cells to reconstitute haematopoiesis. If this progenitor population could successfully sustain multi-lineage haematopoiesis for a prolonged time period, infusion of enhanced progenitor cells regularly could be an alternative option when allogeneic donor is unavailable. Until now, limited but encouraging success has been achieved in this aspect. Ectopic expression of transcription factor Sox17 via retroviruses has been demonstrated to increase the self-renewal potential of multipotent progenitors (MPPs) and therefore conferred on MPPs the potential for long-term multi-lineage reconstitution. 7 Another study has successfully conferred long-term repopulating ability on MPPs with a single miRNA, miR-125a. The enforced expression of miR-125a endowed MPPs with enhanced self-renewal potential, resulting in robust long-term multi-lineage repopulation. 8 NUP98-HOXA10hd fusion gene (NA) has been shown to be potent for haematopoietic stem cells to expand, survive under stress 9,10 and promote engraftment competitiveness. 11

| RNA-Seq and data analysis
MPPs were sorted separately into 200 µL DPBS-BSA buffer (0.5% BSA) using 1.5 mL EP tube. Sequencing libraries of MPPs were generated as previously described for HSC. 11 Briefly, cDNA of sorted 1000 MPPs aliquots was generated, amplified 12  Omnibus public database (GSE146781). Alignment, normalization of genes expression and differential expression genes (DEGs) analysis were performed by RSEM and DESeq2. GO enrichment analysis was performed with clusterProfiler package. Gene set enrichment analysis (GSEA) was performed as described. 13 Heatmaps were plotted using gplots (heatmap.2) and ggplot2 package.
Normal distribution of data was tested with SPSS applying Shapiro-Wilk normality test. The data were represented as mean ± SEM. Two-tailed independent Student's t tests were performed for comparison of two groups of data. P values <.05 were considered statistically significant (*P < .05, **P < .01 and ***P < .001).

| NA-overexpressing MPPs sustain long-term multi-lineage haematopoiesis in primary mice
We have previously established NA-overexpression mouse model and found that ectopic expression of NA confers engraftment competitiveness of LT-HSCs in competitive transplantation assay. 11 We further investigated the cell-context roles of NA in MPPs. We directly sorted 300 MPP (Lin − CD48 − c-kit + Sca1 + CD135 + CD150 − ) cells

| NA upregulates genes regulating long-term haematopoiesis, cell cycle, epigenetic regulation and responses to stress in MPPs
In order to investigate how NA altered MPPs at molecular level, we sorted MPPs from NA and WT mice for transcriptome analysis. Since one thousand cell input for RNA-Seq produced reads uniformly covers all transcripts, 14 we sorted one thousand MPP aliquots for RNA-Seq analysis. Differential gene expression analysis identified 2347 differential expression genes (DEGs) in NA MPPs compared with WT MPPs (Table S1). GO enrichment analysis of upregulated DEGs (>2 fold) using the NA MPPs showed that these DEGs were largely involved in the regulation of cell cycle, epigenetic regulation, responses to stress, RNA splicing and regulation of signalling pathways (Figure 2A). Further gene set enrichment analysis (GSEA) showed NA regulated genes were enriched for gene sets of long-term haematopoiesis and cell cycle ( Figure 2B). Overexpression of Hoxa9 has been reported to expand HSC compartment and results in lympho-myeloid long-term repopulation 15 ; meanwhile, Hoxa9 deficiency impairs the multi-lineage repopulating ability of HSC. 16 Interestingly, NA regulated genes were enriched for Hoxa9_ targets and targets_of_Hoxa9_and_Meis1 ( Figure 2C). Collectively, our data imply that NA endows MPPs with the capability to support long-term haematopoiesis through regulating gene involving in the

| NA-MPPs efficiently generated abundant lineage-committed progenitors
To investigate whether NA-MPPs produce abundant lineage-committed progenitors to sustain long-term haematopoiesis, we inves-  Figure 3G). Taken together, our results suggest that NA MPPs achieve long-term haematopoiesis via generating abundant lineagecommitted progenitors rather than acquiring self-renewal capability.

| D ISCUSS I ON
In this study, we demonstrated that NA-overexpressing MPPs can support long-term multi-lineage haematopoiesis in primary mice.
Few studies have previously reported the robust long-term multilineage reconstitution induced by enhancing self-renewal potential of MPPs in recipient mice. Edyta and colleagues achieved increased self-renewal of MPPs by retroviral ectopic expression of miR125a.
However, development of myeloproliferative disease/leukaemia in secondary and tertiary recipients was observed. 8 Another study successfully employed overexpression of transcription factor Sox17 to confer increased self-renewal of multipotent progenitor cells to support long-term multi-lineage reconstitution. Unfortunately, longterm ectopic expression of Sox17 eventually led to leukemogenesis. 7 In our case, long-term multi-lineage haematopoiesis is unlikely to be induced by enhanced self-renewal potential of MPP, as shown by the absence of LSK population in primary recipient mice and the loss of donor contribution in bone marrow of secondary recipient mice.
In addition, the NA MPP primary recipient mice as well as the secondary mice remained leukaemia free, demonstrating no obvious oncogenesis of our approach. This is consistent with the previous report that the restriction of NUP98-fusion to the homeodomain of In summary, our data show that NA-overexpressing MPPs can support long-term multi-lineage haematopoiesis in primary mice through generating abundant lineage-committed progenitors, which will benefit the application of MPPs in the absence of LT-HSCs to improve clinical outcomes in transplantation settings.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
YD, KW, QW, TW, PZ, XL, YG, LL and HW performed the experiments and analysed data. JD and JW conceived and supervised the study, wrote the manuscript and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon request. All RNA-Seq data are in the GEO database with accession code GSE146781.