Stromal CYR61 Confers Resistance to Mitoxantrone via Spleen Tyrosine Kinase Activation in Human Acute Myeloid Leukaemia

Abstract

Approximately 50% of children with acute myeloid leukaemia (AML) relapse, despite aggressive chemotherapy. The bone marrow stromal environment protects leukaemia cells from chemotherapy (i.e., stroma-induced chemoresistance), eventually leading to recurrence. Our goal is to delineate the mechanisms underlying stroma-mediated chemoresistance in AML. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, which are equally effective in protecting AML cells from chemotherapy-induced apoptosis in AML-stromal co-cultures. We found that CYR61 was highly expressed by stromal cells, and was upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein and is associated with cell-intrinsic chemoresistance in other malignancies. Here, we show that blocking stromal CYR61 activity, by neutralization or RNAi, increased mitoxantrone-induced apoptosis in AML cells in AML-stromal co-cultures, providing functional evidence for its role in stroma-mediated chemoresistance. Further, we found that spleen tyrosine kinase (SYK) mediates CYR61 signalling. Exposure to stroma increased SYK expression and activation in AML cells, and this increase required CYR61. SYK inhibition reduced stroma-dependent mitoxantrone resistance in the presence of CYR61, but not in its absence. Therefore, SYK is downstream of CYR61 and contributes to CYR61-mediated mitoxantrone resistance. The CYR61-SYK pathway represents a potential target for reducing stroma-induced chemoresistance.

Keywords: adhesion; drug resistance; marrow stroma; myeloid leukaemia; signalling.

Fig 1

HS-5 cells secreted many more factors than HS-27A cells. Both parental (pa) and mOrange (mO) stromal cells were cultured for 48 h, and 42 soluble factors were examined in the stroma-conditioned medium by multiplex cytokine assay. Detectable soluble factors are grouped by concentrations (A–D). N = 4–5.

Fig 2

Bone marrow stromal cells conferred resistance to chemotherapy-induced apoptosis in acute myeloid leukaemia (AML) cell lines and primary paediatric patient samples. NB-4 and THP-1 AML cell lines and primary patient samples were kept in suspension, or co-cultured with HS-5 or HS-27A stromal cells, for 24 h (cell lines) or 1 h (patient samples), followed by exposure to chemotherapy drugs for 24 h before Annexin V apoptosis assay. Dose-response apoptosis curves of NB-4 (A1-3), THP-1 (B1-3), a representative patient sample (C1-3) and the averaged results from five patient samples (D1-3) are depicted. N = 4–5. BM, bone marrow mononuclear cells. *, P < 0·05, alone v. HS-5 or HS-27A co-culture; #, P < 0·05, HS-5 v. HS-27A co-culture.

Fig 3

Cell-cell contact and soluble factors differentially contributed to resistance to different chemotherapies in acute myeloid leukaemia (AML) cells. NB-4 and THP-1 AML cells were cultured alone, or co-cultured in Transwell inserts physically separated from stromal cells, and treated with chemotherapy before Annexin V apoptosis assay. Stroma-induced protection of AML cells from etoposide (A1, B1), cytarabine (A2, B2) and mitoxantrone (A3, B3) was shown. N = 3–4. *, P < 0·05, alone v. HS-5, or HS-27A co-culture; +, P < 0 05, alone v. HS-5 co-culture; #, P < 0·05, HS-5 v. HS-27A co-culture.

Fig 4

A subset of stroma-induced genes was commonly upregulated by both stromal cell lines in acute myeloid leukaemia (AML) cells. NB-4 (N) and THP-1 (T) cells were cultured alone, or co-cultured with stromal cells for 48 h, then flow sorted to isolate the mOrange-negative population, from which total RNA was extracted for gene expression profiling. (A) Nineteen genes, including CYR61, were found upregulated in both AML cell lines by both stromal cell lines. The levels of those genes (mean-centred, denoted by gene-specific probesets) are shown. Red/green denotes relatively high/low gene expression, respectively. (B) CYR61 mRNA level was determined by qRT-PCR in NB-4 (N = 4), THP-1 (N = 4), and two AML patient samples cultured alone, or co-cultured with stromal cells. *, P < 0·05, alone v. HS-5 or HS-27A co-culture. (C) CYR61 protein expression was determined by immunocytochemistry in THP-1 cells and primary patient samples cultured alone or co-cultured with HS-5 cells. Representative images of CYR61 (column 1), myeloperoxidase (MPO; AML marker, column 2) staining and merged images of CYR61, MPO, and Hoechst DNA staining for cell nuclei (column 3) are shown. The scale bars shown are 25 μm.

Fig 5

Neutralizing CYR61 reduced stroma-induced mitoxantrone resistance in THP-1 cells. THP-1 and stromal cells were pre-incubated with anti-CYR61 neutralizing antibody or normal rabbit IgG (both at 20 μg/ml) prior to placing in co-culture. After co-culture for 24 h, chemotherapy agent was added, along with anti-CYR61 or control IgG, and apoptosis was measured after another 24 h. The contributions of CYR61 activity to the stroma-induced protection from (A) 1·5 μM etoposide and (B) 50 nM mitoxantrone are shown. N = 3. *, P < 0·05, anti-CYR61 antibody v. normal rabbit IgG.

Fig 6

CYR61 knockdown in stromal cells alleviated stroma-induced mitoxantrone resistance in NB-4 and THP-1 cells. HS-5 and HS-27A cells were transduced with shRNA constructs targeting CYR61. Knockdown efficiency was evaluated by (A) quantitative reverse transcription polymerase chain reaction and (B) Western blotting, respectively. Compared to non-silencing control (NS Ctrl), constructs 2 (C2) and C4 had the highest knockdown efficiency, and these two were used for further functional analyses. The contribution of stromal CYR61 to resistance to 100 nM mitoxantrone (C, D), 1·5 μM etoposide (E, F) or 3 μM cytarabine (G, H) was determined in AML cells co-cultured with C2- or C4-transduced stromal cells in comparison to those co-cultured with NS Ctrl stromal cells. N = 4. *, P < 0·05 as indicated.

Fig 7

Stromal CYR61 upregulated total-SYK (t-SYK) and phospho-SYK (Y348) in acute myeloid leukaemia (AML) cells. T-SYK was determined by flow cytometry in NB-4 (A, N = 3) and THP-1 cells (B, N = 3), cultured alone or co-cultured with non-silencing control (NS Ctrl) HS-27A or CYR61-KD HS-27A cells for 48 h. P-SYK (Y348) was also determined in NB-4 (C, N = 6), THP-1 (D, N = 4) and paediatric AML patient samples (E, N = 9) in the similar culture conditions. The expression levels were quantified by mean fluorescence intensity (MFI). *, P < 0·05, **, P < 0·01, + NS Ctrl v. alone.

Fig 8

Inhibition of p-SYK partially reversed HS-27A-induced mitoxantrone resistance. NB-4 (A, N = 3) and THP-1 cells (B, N = 4) were pre-incubated with SYK inhibitor R406 (30 nM, 300 nM, or 3 μM) or dimethyl sulfoxide (DMSO) vehicle control for 30 min, before co-culture with NS Ctrl HS-27A cells for 48 h. Then p-SYK (Y348) was determined in acute myeloid leukaemia (AML) cells by flow cytometry and quantified by MFI. NB-4 (C, N = 4), THP-1 cells (D, N = 3) and primary AML patient samples (E, N = 5) were pre-incubated with 3 μM R406 or DMSO for 30 min, then were either cultured alone, or co-cultured with NS Ctrl HS-27A or CYR61-KD HS-27A cells. All cells were treated with 300 nM mitoxantrone for 24 h before the Annexin V apoptosis assay. The contribution of SYK activation (Y348) to stromal CYR61-induced resistance to mitoxantrone was depicted. *, P < 0·05 as indicated.