TGF-β Neutralizing Antibody 1D11 Inhibits LIF-JAK-Stat3 Signaling and Enhances Cytarabine Induced Apoptosis in AML Cells in Bone Marrow Microenvironment

Abstract 926

We have previously reported pro-survival effects of TGF-β1 in myelo-monocytic leukemia cells (Xu et al.,Br J Haematol.2008). Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of leukemic BM microenvironment that promote leukemia cell survival and chemoresistance. Our preliminary data indicate that TGF-β neutralizing antibody 1D11 (Genzyme) prevents accumulation of AML cells in a quiescent G0 state under co-culture condition with BM-derived mesenchymal stromal cells (MSC) (Jin et al., ASH abstract 2010). In turn, the chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions.

In this study, we investigated the anti-leukemic effects and molecular mechanisms of action of TGF-β neutralizing antibody 1D11 under hypoxic conditions. We further investigated the anti-leukemic efficacy of 1D11 combined with CXCR4 antagonist plerixafor in the in vivo leukemia models. AML cells (MV4;11 and U937) were propagated under 1% O₂ for at least 14 days to assure their sustained proliferation and survival. Isotype control antibody 13C4 combined with ara-C induced no significant change in apoptosis or cell cycle progression. In MV4;11 cells cultured with 2ng/mL rhTGF-β1, 1D11 (10 μg/mL) induced only minimal apoptosis by itself, yet enhanced low-dose cytarabine (AraC, 0.5 μM) induced apoptosis. This effect was more prominent under hypoxia compared to normoxia (% of subG₁ fraction, 21% O₂: ara-C, 2.6 ± 0.2%, ara-C + 1D11, 10.8 ± 2.5%, p=0.03; 1% O₂: ara-C, 11.3 ± 2.7%, AraC + 1D11, 21.4 ± 0.5%, p=0.001). 1D11 with ara-C abrogated rhTGFβ1-induced accumulation of cells in G₀/G₁ phase (21% O₂; cont, 73.8 ± 4.1, rhTGFβ, 82.2 ± 3.2, rhTGFβ + AraC, 65.4 ± 2.5, rhTGFβ + AraC + 1D11, 50.3 ± 1.9, p=0.001: in 1% O₂; cont, 71.8 ± 1.3, rhTGFβ, 85.4 ± 1.4, rhTGFβ + AraC, 79.3 ± 5.1, rhTGFβ + AraC + 1D11, 67.1 ± 4.0, p = 0.03).

The anti-leukemic efficacy of 1D11 was next examined in an in vivo leukemia model. 1D11 administered at 5 mg/kg IP every other day in combination with ara-C (50 mg/kg IP weekly) decreased leukemia burden of nude mice injected with Baf3/ITD-luciferase leukemia cells (p=0.002). Administration of small molecule CXCR4 inhibitor plerixafor, which successfully diminished cell migration to CXCL12 in vitro, in combination with 1D11 decreased leukemia burden in vivo (p=0.05), and co-administration of ara-C, plerixafor and 1D11 was most effective (bioluminescence intensity, ×10⁷ photons/sec) control, 1.2 ± 0.2; ara-C, 0.94 ± 0.3; plerixafor + 1D11, 0.56 ± 0.1; plerixafor + 1D11 + ara-C, 0.23 ± 0.09, p=0.003).

We next examined the molecular mechanisms responsible for chemosensitization through blockade of TGFβ with 1D11. Treatment with rhTGF-β1 induced upregulation of p21 expression as well as pro-survival phosphorylation of Stat3 in MV4;11 and U937 cells, and these effects were abrogated by 1D11. Knock-down of Stat3 by siRNA increased apoptosis induction in U937 cells cultured in the presence of rhTGFβ1. Notably, 4-fold upregulation of the established TGFβ target, leukemia inhibitory factor (LIF) gene mRNA, was observed after rhTGF-β1 treatment and this was reversed by 1D11. These results indicate that 1D11 inhibits rhTGF-β1-induced autocrine stimulation of pro-survival LIF-JAK-Stat3 signal transduction pathway in AML cells.

In summary, blockade of TGF-β by 1D11, and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment. These findings warrant further investigations in human clinical trials.