p53 activation of mesenchymal stromal cells partially abrogates microenvironment-mediated resistance to FLT3 inhibition in AML through HIF-1α-mediated down-regulation of CXCL12

Abstract

Fms-like tyrosine kinase-3 (FLT3) inhibitors have been used to overcome the dismal prognosis of acute myeloid leukemia (AML) with FLT3 mutations. Clinical results with FLT3 inhibitor monotherapy have shown that bone marrow responses are commonly less pronounced than peripheral blood responses. We investigated the role of p53 in bone marrow stromal cells in stromal cell-mediated resistance to FLT3 inhibition in FLT3 mutant AML. While the FLT3 inhibitor FI-700 induced apoptosis in FLT3 mutant AML cells, apoptosis induction was diminished under stromal coculture conditions. Protection appeared to be mediated, in part, by CXCL12 (SDF-1)/CXCR4 signaling. The protective effect of stromal cells was significantly reduced by pre-exposure to the HDM2 inhibitor Nutlin-3a. p53 activation by Nutlin-3a was not cytotoxic to stromal cells, but reduced CXCL12 mRNA levels and secretion of CXCL12 partially through p53-mediated HIF-1α down-regulation. Results show that p53 activation in stroma cells blunts stroma cell-mediated resistance to FLT3 inhibition, in part through down-regulation of CXCL12. This is the first report of Nutlin effect on the bone marrow environment. We suggest that combinations of HDM2 antagonists and FLT3 inhibitors may be effective in clinical trials targeting mutant FLT3 leukemias.

Figure 1

MSCs protect FLT3/ITD AML cells from FI-700- and doxorubicin-induced cell death. (A) FLT3/ITD MOLM-13 cells were treated with 800nM FI-700 (FI), 2μM Nutlin-3a (N3a), 100nM doxorubicin (Dox), 200nM AraC or left untreated and cultured for 24 hours in the presence or absence of MSCs from 3 normal subjects, and the numbers of viable cells and annexin V–positive fractions were measured. (B-C) MOLM-13 cells were treated for 72 hours with the indicated concentrations of FI-700 (B) or doxorubicin (C) in the presence or absence of MSCs, and the numbers of viable cells and annexin V–positive fractions were measured. Asterisk (*) indicates significance at P < .05.

Figure 2

p53-activated MSCs exhibit reduced protection of FLT3/ITD AML cells from FI-700–induced apoptosis. (A) MSCs were pretreated with 10μM Nutlin-3a (N3a) for 24 hours. After the wells were washed 3 times with MEM-α medium (control medium: Ctrl-Med), MOLM-13 cells were treated with 800nM FI-700 for 24 hours in the presence or absence of MSCs from 3 normal subjects (N-MSC) or 3 AML patients (AML-MSC). Asterisk (*) indicates significance at P < .05 (1-way ANOVA/Tukey). (B) MSCs were transfected with either control (SiC) or p53 siRNA (Sip53). Twenty-four hours posttransfection, cells were subsequently treated with 10μM Nutlin-3a (N3a). After washing, MOLM-13 cells were treated with 800nM FI-700 for 24 hours. Intensity of the immunoblot signals was quantified and the relative intensity of p53 compared with β-actin was calculated.

Figure 3

Nutlin-pretreated MSCs exhibit reduced protection of FLT3/ITD AML cells from FI-700–induced mitochondrial apoptosis. Normal MSCs (N-MSC) were pretreated with 10μM Nutlin-3a (N3a) for 24 hours. MOLM-13 cells were treated with 800nM FI-700 for 24 hours in the presence or absence of MSCs. Bax conformational change (A) and activation of caspase-3 (B) were analyzed by flow cytometry. Representative flow cytometry results are also shown. Asterisk (*) indicates significance at P < .05 (1-way ANOVA/Tukey). (C) Mcl-1, Bcl-XL and survivin levels were determined by Western blotting. Intensity of the immunoblot signals was quantified and the relative intensity compared with β-actin was calculated.

Figure 4

MSCs protect MOLM-13 cells by production of soluble factors, regulated by p53 activation. (A) Normal MSCs (N-MSC) were pretreated with 10μM Nutlin-3a (N3a) for 24 hours. MOLM-13 cells were treated with 800nM FI-700 for 24 hours in the presence or absence of normal MSCs. Cells were collected from upper compartment when culture inserts were used. NS indicates not significant. (B) MOLM-13 cells were treated with the indicated concentrations of FI-700 in MEM-α medium (control medium), MSC-conditioned medium (conditioned medium) or with normal MSCs. Asterisk (*) indicates significance at P < .05 (1-way ANOVA/Tukey).

Figure 5

Nutlin-3a treatment reduces CXCL12 expression in MSCs and may affects CXCL12/CXCR4 signaling in AML cells. (A) MSCs from 3 normal subjects were treated for 24 hours with 10μM Nutlin-3a, and transcripts were quantitated by real-time PCR. Each real-time PCR was performed in duplicate, and the average fold induction relative to untreated cells is shown. (B) MSCs from 5 normal subjects (N-MSC) were cultured for 48 hours in the presence or absence of 10μM Nutlin-3a, and CXCL12 concentrations in the culture medium were determined. Results are expressed as mean ± SEM (C-D) MOLM-13 cells were treated with 800nM FI-700, 1 ng/mL CXCL12 and 200μM AMD3100 for 24 hours in the presence or absence of MSCs, and annexin V–positive fractions were measured. In some cases, MSCs were pretreated with 10μM Nutlin-3a (N3a) for 24 hours. Asterisk (*) indicates significance at P < .05.

Figure 6

Nutlin-3a abrogates MSC-mediated resistance to FI-700 in primary AML cells. Fifteen FLT3 mutant samples and 15 FLT3 wild-type samples were treated for 72 hours with 800nM FI-700 in the presence or absence of MSCs. Annexin V–positive fractions were determined. MSCs were either Nutlin-3a–pretreated or untreated. In each patient sample, the percentage of annexin V–positive cells in control medium (spontaneous apoptosis) was set as “0” (control) and the extent of apoptosis was expressed as percent changes from this baseline. Results are expressed as mean ± SEM. Asterisk (*) indicates significance at P < .05.

Figure 7

Nutlin-3a reduces CXCL12 mRNA levels in MSCs partially through the HIF-1α pathway. MSCs were treated with 10μM Nutlin-3a for 24 hours in the presence or absence of 0.1 μg/mL doxycycline. Intensity of the immunoblot signals was quantified and the relative intensity compared with β-actin was calculated. The bar graphs show the ratio of the CXCL12 mRNA level in Nutlin-treated cells compared with that in Nutlin-untreated cells. Asterisk (*) indicates significance at P < .05. HI indicates high intensity image.