TGF-β-Neutralizing Antibody 1D11 Enhances Cytarabine-Induced Apoptosis in AML Cells in the Bone Marrow Microenvironment

Abstract

Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of the leukemic BM microenvironment in promoting leukemia cell survival and chemoresistance. High levels of transforming growth factor beta 1 (TGFβ1) produced by BM stromal cells in the BM niche regulate cell proliferation, survival, and apoptosis, depending on the cellular context. Exogenous TGFβ1 induced accumulation of acute myeloid leukemia (AML) cells in a quiescent G0 state, which was further facilitated by the co-culture with BM-derived mesenchymal stem cells (MSCs). In turn, TGFβ-neutralizing antibody 1D11 abrogated rhTGFβ1 induced cell cycle arrest. Blocking TGFβ with 1D11 further enhanced cytarabine (Ara-C)-induced apoptosis of AML cells in hypoxic and in normoxic conditions. Additional constituents of BM niche, the stroma-secreted chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. Treatment with 1D11 combined with CXCR4 antagonist plerixafor and Ara-C decreased leukemia burden and prolonged survival in an in vivo leukemia model. These results indicate that blockade of TGFβ by 1D11 and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment.

Figure 1. TGF-β-neutralizing antibody1D11 suppresses TGF-β signaling in AML cells.

(A) Percentage of MV4;11, U937 and THP-1 cells showing annexin V positivity after 72 hours of treatment with or without rhTGF-β1 (2 ng/ml) and the indicated concentrations of Ara-C. Graphs show the means ± SD of the results from three independent experiments. (B)(C) MV4;11 and U937 cells were treated with TGF-β1 (2 ng/ml), 1D11 (10 µM), or 13C4 (10 µM) for 24 hours. (B) Expression of phospho (p-) Smad2 and p21 proteins by western blot analysis. Clarified lysates were probed with antibodies to p-Smad2, p21 and α-tubulin. Results shown are representative of three independent experiments. (C) PAI-1 (plasminogen activator inhibitor-1) mRNA expression detected by TaqMan RT-PCR analysis. The abundance of transcripts of PAI-1 relative to the abundance of transcripts of GAPDH was determined as described in Materials and Methods. Results shown are representative of three experiments. *P<0.05, ** P<0.01.

Figure 2. 1D11 reverses TGF-β-mediated cell cycle inhibition and anti-apoptotic effects.

(A)(B) MV4;11 cells were treated with rhTGF-β1 (2 ng/ml), with and without 1D11 (10 µM) or 13C4 (10 µM), for 72 hours under serum-starved conditions and cultured without and with MSCs, as described in Materials and Methods. (A) The percentage of Annexin V–positivity by flow cytometry analysis. Bar graph represent percentages of annexin V–positive cells (mean± SD) from three independent experiments. (B) Flow cytometric cell cycle analysis by PI staining. Bar graphs represent percentages of SubG₁- and G₀/G₁-phase cells (mean± SD) from three independent experiments. Graphs show the means ± SD of the results from three independent experiments. *P<0.05, **P<0.01. (C) Primary AML cells harboring mutant FLT3-ITD (Pt #1–3) or wt-FLT3 (Pt #4–10) were incubated for 48 h with rhTGF-β1 (2 ng/ml), 1D11 (10 µM), with or without MSCs co-culture condition. Clinical characteristics of patients are summarized in Table S1. The percentage of G₀/G₁-, S- and G₂/M-phase cells were detected by flow cytometric PI cell cycle analysis. Average show the means ± SEM of the results from ten (i) or six (ii) primary AML cells, respectively.

Figure 3. 1D11 enhances the proapoptotic effect of Ara-C in both normoxic and hypoxic conditions.

MV4;11 cells were treated with Ara-C (0.5 µM), rhTGF-β1 (2 ng/ml), 1D11 (10 µM), and/or 13C4 (10 µM) for 72 hours under normoxic (A) and hypoxic (B) conditions. Percentages of apoptotic cells (annexin V–positive cells) were analyzed by flow cytometry. Graphs show the means ± SD of the results from three independent experiments. *P<0.05, **P<0.01.

Figure 4. Plerixafor and 1D11 inhibit CXCL12-induced migration.

Chemotaxis assay of MV4;11 cells treated with TGF-β1 (2 ng/ml), 1D11 (10 µM), and/or plerixafor (1 µM) for 18 hours. Cells were stimulated with CXCL12 (100 ng/mL) for 4 hours. In all experiments, the chemotactic index was calculated as described in Materials and Methods. Graphs show the means ± SD of the results from three independent experiments. *P<0.05, **P<0.01.

Figure 5. Combination of 1D11, Plerixafor, and Ara-C induces potent antitumor effects in vivo .

Ba/F3-ITD-luciferase leukemia cells were injected into nude mice as described in Materials and Methods. (A) Serial bioluminescence images (i) and luciferase intensity (ii) of mice in the groups receiving 1D11, Ara-C, Plerixafor, one of the combinations indicated, or no treatment (control) were taken on days 13 and 17 after tumor cell injection. *P≤0.05, **P<0.01. (B) Histologic sections of bone marrow taken from mice on day 17 were stained with H&E or anti-GFP antibody.

Figure 6. Simultaneous blockade of TGF-β and CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment.

Within the BM microenvironment, TGF-β secreted by stromal cells induces cell cycle arrest of AML cells through p21 upregulation that is inhibited by 1D11. Combination of Plerixafor, which inhibits CXCL12-induced migration, and 1D11, which blocks TGF-β signaling, abrogates stroma-mediated chemoresistance and promotes the antileukemia effects of chemotherapy.