Mesenchymal niche remodeling impairs hematopoiesis via stanniocalcin 1 in acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) disrupts the generation of normal blood cells, predisposing patients to hemorrhage, anemia, and infections. Differentiation and proliferation of residual normal hematopoietic stem and progenitor cells (HSPCs) are impeded in AML-infiltrated bone marrow (BM). The underlying mechanisms and interactions of residual hematopoietic stem cells (HSCs) within the leukemic niche are poorly understood, especially in the human context. To mimic AML infiltration and dissect the cellular crosstalk in human BM, we established humanized ex vivo and in vivo niche models comprising AML cells, normal HSPCs, and mesenchymal stromal cells (MSCs). Both models replicated the suppression of phenotypically defined HSPC differentiation without affecting their viability. As occurs in AML patients, the majority of HSPCs were quiescent and showed enrichment of functional HSCs. HSPC suppression was largely dependent on secreted factors produced by transcriptionally remodeled MSCs. Secretome analysis and functional validation revealed MSC-derived stanniocalcin 1 (STC1) and its transcriptional regulator HIF-1α as limiting factors for HSPC proliferation. Abrogation of either STC1 or HIF-1α alleviated HSPC suppression by AML. This study provides a humanized model to study the crosstalk among HSPCs, leukemia, and their MSC niche, and a molecular mechanism whereby AML impairs normal hematopoiesis by remodeling the mesenchymal niche.

Keywords: Adult stem cells; Hematology; Hematopoietic stem cells; Leukemias; Stem cells.

Figure 1. AML induces quiescence and prevents differentiation in normal HSPCs.

(A–G) CD34⁺ cells cocultured with MSCs alone (CD34⁺ alone) (n = 4–7) or +AML cell lines (n = 3–7) or +AML primary patient samples (n = 3–7). After 4 days of coculture, CD34⁺ cells were plated for CFU or LTC-IC assays or implanted into NSG mice. (B) Cell counts of total non-leukemic hematopoietic cells. AML patient samples: AML1–4. (C) Representative FACS plots of cell cycle analysis of CD34⁺ cells based on DAPI and Ki-67 staining. (D) Quiescent (Ki-67^–DAPI^–) cells within normal progenitors (CD34⁺CD38⁺) and HSPCs (CD34⁺CD38^–). AML1, -2, -5, -8, and -9. (E) Proportions of normal HSPCs within CD34⁺ cells. AML1-5, -8, and -9. (F) Engraftment in primary NSG recipients. Three independent experiments with 1–7 mice/group per experiment. (G) Secondary recipients. AML1–3. Three independent experiments with 2–4 mice/group per experiment. (H–K) Collagen scaffolds seeded with MSCs were injected with CB CD34⁺ cells alone or +GFP⁺ AML cell lines (n = 4) and transplanted into NSG-SGM3 recipients. In the case of AML patient samples (n = 5–8), the CB CD34⁺ cells were either HLA-A2 mismatched or transduced to express GFP. Scaffold retrieval: 2–3 weeks (+AML cell lines) or 5–8 weeks (+AML patient samples). EdU i.p. injection: 16 hours prior to scaffold retrieval. (I) Non-leukemic human CD45⁺ hematopoietic cells per scaffold. AML1–5 and 11–13. Twelve to 26 scaffolds in 6–10 mice/group. (J) EdU incorporation in non-leukemic human CD45⁺ hematopoietic cells. AML1–5. Twelve to 26 scaffolds in 5–7 mice/group. (K) Proportions of CD34⁺ HSPCs within non-leukemic human CD45⁺ hematopoietic cells. Five to 11 mice/group. AML1, -3, and -4. Each AML cell line or patient sample is represented as a unique symbol. CD34⁺ cells alone were used as control and for normalization. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Mann-Whitney U test (B and D–G), Kruskal-Wallis with Dunn’s (I and J), and Wilcoxon’s matched-pairs signed-rank test (K).

Figure 2. AML induces HSPC quiescence via mesenchymal niche–secreted factors.

(A–C) CD34⁺ cells were cultured alone or with AML cell lines with 100 ng/mL SCF/FLT-3L and 20 ng/mL TPO + 10% FBS with (+) and without (–) MSCs. CD34⁺ cells alone were used as control and reference for normalization. Cell counts of CD34⁺ cells after 4 days (B) and CTV^bright cells among CD34⁺ cells (C). (D and E) MSCs were cultured alone (n = 6) or cocultured with AML cell lines expressing iCASP9 (n = 5) for 5 days. AP1903 (5 nM) was added on day 4, inducing apoptosis and allowing the removal of AML. CB CD34⁺ cells were then added in fresh medium to the preconditioned MSCs for 48 hours. (E) Percentage of CTV^bright cells retrieved after 48 hours. (F) CD34⁺ cells cocultured with MSCs and AML cell lines (n = 3) directly or separated from MSCs and AML via a 0.4-μm Transwell insert. (G) CTV^bright cells among CD34⁺ cells. (H) Gene expression analysis of MS-5 cells after coculture for 7 days with AML cell lines (n = 7), AML patient samples (n = 2), or normal CD34⁺ cells (n = 3) as control. Heatmap of shared upregulated secreted factors with logarithmic fold change (logFC) > 1. FC, fold change. Data are presented as mean ± SEM, with each AML cell line or patient sample as a unique symbol (E) or as box-and-whisker plots, with bounds from 25th to 75th percentile, median line, and whiskers ranging from smallest to largest values of 4 measurements from CD34⁺ alone or +AML cell lines (n = 3–4) (B, C, and G). *P < 0.05,**P < 0.01, ***P < 0.001, ****P < 0.0001 by Kruskal-Wallis test with Dunn’s test (B, C, and G) and Mann-Whitney U test (E).

Figure 3. MSC-secreted STC1 is upregulated in AML patient plasma.

(A) mRNA expression of STC1 in MSCs cultured alone (n = 4), +CB CD34⁺ cells (n = 4), or +AML cell lines (n = 3)/patient samples (n = 3) for 5 days; measured by quantitative PCR (qPCR) and normalized to MSCs cultured alone (n = 4). AML patient samples: AML6, -8, and -9. (B) STC1 protein in supernatant of MSCs cocultured with CD34⁺ cells, +AML cell lines (n = 7)/patient samples (n = 8); measured by ELISA and normalized to MSCs + CD34⁺ cells (CD34⁺ alone). AML1–5, -8, -10, and -11. STC1 protein was measured by ELISA (C) in AML patient BM plasma from nonmatched diagnosis (n = 27) and remission (n = 21) samples and normalized to remission and (D) from 5 matched diagnosis/remission samples. (E) STC1 protein in AML patient PB plasma at diagnosis (n = 26) and healthy donors (n = 20). (F) Correlation of STC1 protein concentration between BM and PB plasma (n = 13). (G) Correlation of platelet (PLT) count in PB and STC1 protein in PB plasma from AML patients at diagnosis (n = 26). Data are presented as mean ± SEM, with each patient sample as a dot (C–G) or unique symbol (A and B). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Kruskal-Wallis test with Dunn’s test (A and B), Mann-Whitney U test (C and E), Wilcoxon’s matched-pairs signed-rank test (D), and Pearson’s correlation and linear regression test (F and G).

Figure 4. STC1 induces reduction of CB and BM HSPC proliferation ex vivo.

(A and B) MSCs were cocultured with CB CD34⁺ cells and supplemented with rSTC1 or PBS (vehicle control) for 5 days. (B) Quantification of cell count. (C–F) Results of GSEA of HSPCs (CD34⁺CD38^–) cocultured for 48 hours with MSCs and supplemented with rSTC1 or PBS (vehicle control) (n = 3). (G) Proportions of Ki-67^– quiescent cells in CB CD34⁺ cells treated with rSTC1 and PBS. (H) LTC-IC frequency in sorted CD34⁺ cells. (I–L) MSCs were cocultured with BM CD34⁺ from 2 donors and supplemented with rSTC1 or PBS (vehicle control) for 5 days. (I) Total expansion of hCD45⁺ cells relative to day 0. Quantification of (J) HPCs and HSPCs normalized to PBS on day 5 and (K) proportions of HPCs/HSPCs that had undergone 0–1 divisions normalized to PBS. (L) LTC-IC frequency per CD34⁺ cells. Data are presented as mean ± SEM, n = 4–7. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed Student’s t test. NES, normalized enrichment score.

Figure 5. STC1 induces reduction of HSPC proliferation in AML in vivo.

(A–C) MSC scaffolds with normal CD34⁺ cells were implanted into NSG-S mice. rSTC1 was injected every other day for 10 days starting on day 2 after implantation. Three mice/group with 4 scaffolds per mouse. (B) CTV⁺ cells among total human CD45⁺ and CD34⁺ cells. (C) LTC-IC frequency of sorted human CD45⁺lineage^– cells. (D–F) MSC scaffolds with normal CD34⁺ cells and AML cell lines (U937, OCI-AML3) were implanted into NSG-S mice. IgG or anti-STC1 antibody was injected subcutaneously every other day for 2 weeks. Seven mice/group with 2–4 scaffolds per mouse. (E) Absolute cell counts of AML and normal human CD45⁺ cells per mouse. (F) CTV^bright cells among CD34⁺ cells and normalized to CD34⁺ cells alone. Each dot represents data from 2–6 pooled scaffolds implanted in 1 recipient. *P < 0.05 by 2-tailed paired Student’s t test (B, C, and F) or Wilcoxon’s matched-pairs signed-rank test (E).

Figure 6. HIF-1α stabilization in MSCs induces STC1 secretion in AML.

(A) Common gene ontology of upregulated genes from MSCs cocultured with AML cell lines or patient samples. logFE, logarithmic fold expression. (B) mRNA expression of hypoxia-regulated genes in MSCs. Data were obtained from 2–9 measurements of MSCs cultured alone at 20% O₂ (normoxia) (n = 9) or 3% O₂ (hypoxia) (n = 2), or cocultured with CB CD34⁺ cells (n = 4) or AML cell lines (n = 3)/patient samples (n = 4) for 5 days at 20% O₂; measured by qPCR and normalized to MSCs (normoxia). AML patient samples: AML6–9. Measurements from AML cell lines, n = 3–4. (C) Immunoblot of HIF-1α and β-actin (loading control) of whole cell lysate of MSCs cultured alone or with AML patient samples. AML1, -3, and -7. (D–F) MSCs were lentivirally transduced to express shRNA against dsRed fluorescent protein (RFP; shCTL) or an shHIF-1α construct. (D) mRNA expression of hypoxia-regulated genes in cocultured MSCs normalized to shCTL-MSCs + CD34⁺ cells alone. AML7. n =2. (E) STC1 secretion in supernatant of MSCs + CD34⁺ cells alone or +AML5, -7, and -8. n = 3. (F) STC1 mRNA expression in transduced MSCs sorted from pooled scaffolds with CB CD34⁺ alone or +AML cell line U937. Four mice/group with 2 scaffolds per mouse. Data are presented as mean ± SEM, except in D, which shows mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by Kruskal-Wallis test with Dunn’s test (A), 2-tailed Student t test corrected with the Holm-Šidák method (D and E), and ANOVA with Tukey’s test (F).

Figure 7. HIF-1α knockdown in MSCs partially rescues HSPC proliferation in AML.

(A–C) MSCs were lentivirally transduced to express the shCTL or shHIF-1α construct and cocultured with CD34⁺ cells alone or AML cell lines with PBS or rSTC1. CD34⁺ cells alone were used as control and reference for normalization. Data are presented as box-and-whisker plots, with bounds from 25th to 75th percentile, median line, and whiskers ranging from smallest to largest values of 3 measurements per AML cell line (KG1A, OCI-AML3, U937, ML1, MOLM-13). (B) CTV^bright cells among CD34⁺ cells. (C) Proportions of normal HSPCs among normal CD45⁺ cells. (D–F) MSCs were lentivirally transduced to express shRNA against RFP (shCTL) or shHIF-1α construct, and seeded in scaffolds before the injection of normal CD34⁺ cells alone or with the AML cell line U937. Each dot represents data from 1 scaffold. Five mice/group with 2–4 scaffolds per mouse. (E) Cell counts of total normal CD45⁺ cells. (F) Proportions of lineage^–CD34⁺ cells among normal CD45⁺ cells. (G) Proportions of EdU⁺ cells among normal CD45⁺ cells. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Friedman’s test with Dunn’s test (B and C) or Mann-Whitney U test (E–G).