Mesenchymal COX2-PG secretome engages NR4A-WNT signalling axis in haematopoietic progenitors to suppress anti-leukaemia immunity

Abstract

The bone marrow (BM) microenvironment (niche) plays important roles in supporting normal/abnormal haematopoiesis. We investigated the interaction between leukaemic mesenchymal niche and haematopoietic stem and progenitor cells (HSPCs) using the model of Fanconi anaemia (FA), a genetic disorder characterized by BM failure and leukaemia. Healthy donor HSPCs co-cultured on mesenchymal stromal cells (MSCs) derived from FA patients with acute myeloid leukaemia (AML) exhibited higher human engraftment and myeloid expansion in Non-obese diabetic severe combined immunodeficiency IL-2γ^-/- /SGM3 recipients. Untargeted metabolomics analysis revealed the progressively elevated prostaglandins (PGs) in the MSCs of FA patients with myelodysplastic syndromes (MDS) and AML. Reduced secretion of PGs subsequent to inflammatory cyclooxygenase 2 (COX2) inhibition ameliorated HSPC/myeloid expansion. Transcriptome analysis demonstrated dysregulation of genes involved in the NR4A family of transcription factors (TFs) and WNT/β-catenin signalling pathway in FA-AML-MSC-co-cultured-CD34⁺ cells. COX2 inhibition led to significantly decreased NR4A TFs and WNT signalling genes expression. Mechanistically, NR4A1 and NR4A2 synergistically activate the CTNNB1 gene promoter . Knocking down CTNNB1 or NR4A1 in AML-MSC-co-cultured-CD34⁺ cells increased leukaemia-reactive T-effector cells production and rescued anti-leukaemia immunity. Together, these findings suggest that specific interactions between leukaemic mesenchymal niche and HSPCs orchestrate a novel COX2/PG-NR4A/WNT signalling axis, connecting inflammation, cellular metabolism and cancer immunity.

Keywords: WNT signalling; haematopoietic stem cell transplantation; mesenchymal stromal cells; nuclear hormone transcription factors; prostaglandins.

Fig 1.

NSGS mice transplanted with normal cells co-cultured on FA-AML MSCs exhibit haematopoietic stem and progenitor cells and myeloid expansion. 5 × 10⁵ normal bone marrow (BM) cells co-cultured on mesenchymal stromal cells (MSCs) from healthy donors (HD), Fanconi anaemia (FA) patients with cytopenias but no cancer (FA-BMF) or FA patients with acute myeloid leukaemia (FA-AML) (3–5 BM samples for each group) were transplanted into sublethally-irradiated NSGS mice. Recipients were intraperitoneally (i.p.) injected with the GVHD inhibitor OKT3 (10 μg/kg) at 24 h and 1 week after transplant. BM cells were subjected to flow cytometry analysis for human cell content at 12 weeks post-transplant. (A) Representative flow cytometry plots of total human engraftment (hCD45⁺; left), CD34⁺ (middle) and myeloid (CD33⁺)/lymphoid (CD19⁺) cells. (B) Quantification of human cell content by flow cytometry analysis depicted in (A). Results are means ± SD of three independent experiments (n = 9–12 for each group). **P < 0·01. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig 2.

Elevated levels of prostaglandins (PGs) in FA-MDS and FA-AML MSCs. Liquid chromatography-mass spectroscopy -based untargeted metabolomics analysis of mesenchymal stromal cells (MSCs) from healthy donors (HD), Fanconi anaemia (FA) patients with cytopenias but no cancer (FA-BMF), FA patients with myelodysplastic syndrome (FA-MDS) or FA patients with acute myeloid leukaemia (FA-AML). (A) Cloud plot presentation of metabolite features of FA-MDS versus HD MSCs and FA-AML versus HD MSCs with fold change ≥3 and P ≤ 0·01. The statistical significance of the fold change was calculated by a Welch t test with unequal variances. Upregulated features (features that have a positive fold change) are graphed above the x-axis in green while downregulated features (features that have a negative fold change) are graphed below the x-axis in red. The x-axis represents retention time. The y-axis represents mass-to-charge (m/z) ratio. Features with higher fold change have larger radii. Features with lower P-value have higher colour intensity. (B) Venn diagram demonstrating the separate and overlapping metabolite features in BMF, MDS and AML MSCs compared to HD MSCs showing both upregulated and downregulated metabolites with fold change ≥3 and P value ≤0·01. (C) The levels of the metabolites in the Arachidonic acid metabolism pathway are shown. (D) Elevated level of the indicated PGs in MSCs analysed by enzyme-linked immunosorbent assay. Results are means ± SD of three independent experiments (n = 9 for each group). *P < 0·05; **P < 0·01.

Fig 3.

The AML-MSC COX2-PG secretome promotes HSPC/myeloid expansion in humanized recipients. (A) Schematic representation of the experimental design. Reduction of prostaglandin (PG) biosynthesis by COX2 inhibitor (COX2i) in mesenchymal stromal cells (MSCs) from Fanconi anaemia (FA) patients with acute myeloid leukaemia (FA-AML). FA-AML MSCs were pre-treated with COX2i (celecoxib, 10 μmol/l) for 2 h followed by co-culture with normal bone marrow (BM) CD34⁺ cells for two weeks. The progenies were then transplanted into sublethally-irradiated NSGS recipients. In another set of experiments, normal BM CD34⁺ cells were co-cultured on healthy donor (HD) MSCs for 2 weeks followed by BM transplantation into NSGS mice. The recipients were then injected with 16–16 dimethyl-PGE₂ (dmPGE₂; 10 μg/kg body weight) twice per day for 7 days starting at 24 h after transplantation. (B) COX2i celecoxib reduces PG production in FA-AML MSCs. FA-AML MSCs were pre-treated with COX2i (COX2i; celecoxib, 10 μmol/l) or vehicle (5% dimethyl sulphoxide) for 2 h. The levels of the indicated PGs in MSCs were measured by enzyme-linked immunosorbent assay. Results are means ± SD of three independent experiments (n = 9 for each group). (C) COX2i celecoxib prevents haematopoietic stem and progenitor cell (HSPC)/myeloid expansion of FA-AML MSC-co-cultured cells. Co-cultured cells described in (B) were transplant into sublethally-irradiated NSGS mice (5 × 10⁵ cells/mouse; n = 6). Total human engraftment (hCD45⁺), HSPCs (CD34⁺) and myeloid (CD33⁺)/lymphoid (CD19⁺) cells were analysed by flow cytometry eight weeks post-transplant. Results are means ± SD of three independent experiments (n = 9–12 for each group). (D) dmPGE₂ treatment induces HSPC/myeloid expansion in NSGS recipients. The NSGS recipient mice transplanted with HD MSC co-cultured cells were subjected to daily i.p. injection of dmPGE₂ (10 μg/kg body weight) or vehicle (Neat oil) twice per day for 7 days starting at 24 h post-transplant. Total human engraftment (hCD45⁺), HSPCs (CD34⁺) and myeloid (CD33⁺)/lymphoid (CD19⁺) cells of the recipients were analysed by flow cytometry. Results are means ± SD of three independent experiments (n = 9–12 for each group). *P < 0·05, **P <0·01. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig 4.

The link between AML-MSC COX2-PG secretome and NR4A-Treg signalling. (A) Mesenchymal inhibition of COX2 reduces the secretion of prostaglandins (PGs). Mesenchymal stromal cells (MSCs) from Fanconi anaemia (FA) patients with acute myeloid leukaemia (FA-AML) were pre-treated with COX2 inhibitor (celecoxib, 10 μmol/l) or vehicle (5% dimethyl sulphoxide) for 2 h, and the levels of the indicated PGs in the culture medium were measured by enzyme-linked immunosorbent assay. Results are means ± SD of three independent experiments (n = 9 for each group). (B) Mesenchymal inhibition of COX2 reduces the expression of the NR4A transcription factors (TFs) in co-cultured CD34⁺ cells. The MSCs in (A) were co-cultured with normal CD34⁺ cells for 2 weeks and expression of the three NR4A TF genes was determined by real-time polymerase chain reaction (PCR). Results are means ± SD of three independent experiments (n = 9 for each group). (C) Mesenchymal inhibition of COX2 reduces the expression of Treg genes in co-cultured CD34⁺ cells. The MSCs in (A) were co-cultured with normal CD34⁺ cells for 2 weeks and expression of FOXP3 and CTLA4 was determined by real-time PCR. The normal CD34⁺ cells were from 12 healthy donors. Results are means ± SD of three independent experiments (n = 9 for each group). (D) 16–16 dimethyl-PGE₂ (dmPGE₂) treatment increases the expression of the NR4A TF genes in co-cultured CD34⁺ cells. MSCs derived from HD were pre-treated with dmPGE₂ followed by co-culture with normal CD34⁺ cells for 2 weeks. The expression of the three NR4A TF genes was determined by real-time PCR. Results are means ± SD of three independent experiments (n = 9 for each group). (E) dmPGE₂ treatment increases the expression of Treg genes in co-cultured CD34⁺ cells. Co-cultured normal CD34⁺ cells described in (D) were subjected to real-time PCR analysis for FOXP3 and CTLA4. The normal CD34⁺ cells were from 12 healthy donors. Samples were normalized to the level of GAPDH mRNA. Results are means ± SD of three independent experiments (n = 9 for each group). *P < 0·05, **P < 0·01. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig 5.

Crosstalk between NR4A and WNT signalling in regulation of anti-leukaemia T effector cells. (A) NR4A1 and NR4A2 act synergistically to enhance CTNNB1 reporter activity. HEK293 cells expressing a CTNNB1 reporter construct containing −591 to +147 of the proximal CTNNB1 promoter were co-transfected with the indicated combinations of the NR4A transcription factors (TFs). Twenty-four hours after transfection, cells were analysed for luciferase activity. Results are means ± SD of three independent experiments (n = 9 for each group). (B) Enhanced WNT activation in CD34⁺ cells co-cultured with mesenchymal stromal cells (MSCs) from Fanconi anaemia (FA) patients with acute myeloid leukaemia (FA-AML). CD34⁺ cells were transduced with 7TGC-eGFP WNT reporter lentivirus followed by co-culture with HD-MSC or FA-AML-MSC for two weeks. Percentage of GFP⁺ cells were determined by Flow cytometry. Results are means ± SD of three independent experiments (n = 9 for each group). (C) CTNNB1 or NR4A1 knockdown reduces CD8⁺CCR7⁺ T cells. Normal CD34⁺ cells were transfected with 25 nmol/l siGenome SMARTpool siRNAs for CTNNB1, NR4A1 or non-targeted Scramble. After 48 h, cells were co-cultured with FA-AML MSCs for 2 weeks and subjected to by flow cytometry analysis for different T-cell subsets. The graphs show quantification of the indicated T-cell subsets in gated CD3CD4 T cells (CD25⁺FOXP3⁺; left) or gated CD3CD8 T cells (CD8⁺CCR7⁺; right). Results are means ± SD of three independent experiments (n = 9 for each group). (D, E) CTNNB1 or NR4A1 knockdown rescues anti-leukaemia immunity. CD8⁺ or CD8⁺CCR7⁺ cells were sorted by FACS. The indicated T-cell subsets or non-sorted cells expressing Scramble shRNA or shRNA targeting CTNNB1 (D) or NR4A1 (E) were then plated with bone marrow cells from HLA-mismatched AML patients in a 4 h cytotoxicity assay at Effector:Target (E:T) ratios from 20:1 to 2–5:1. Data were pooled from two separate experiments, and six replicate wells were used for each dilution of effector cells. Data are displayed as mean specific lysis ± SD. Results are means ± SD of three independent experiments (n = 9 for each group). *P < 0·05, **P < 0·01. [Colour figure can be viewed at wileyonlinelibrary.com]