Abstract 3543

Transforming growth factor β (TGF-β) is an essential regulator of cell proliferation, survival, and apoptosis, depending on the cellular context. We have previously reported pro-survival effects of TGF-β1 in myelo-monocytic leukemia cells (Xu et al., Br J Haematol.2008) and the anti-leukemic effects of action of TGF-β neutralizing antibody under TGF-β abundant and hypoxic bone marrow (BM) microenvironment (ASH, 2012). Friend leukemia virus integration 1 (FLI-1), a member of Ets transcriptional factors, plays a pivotal role in the regulation of extracellular matrix (ECM) genes, and is known to be negatively regulated through TGF-β1-dependent acetylation. We have recently reported that abnormal (high or low) expression of FLI-1 protein analyzed by reverse phase protein arrays is associated with inferior remission duration and reduced survival (Kornblau et al., Blood, 2011). Notably, among 195 proteins tested, FLI-1 expression correlated most with SMAD4, the common mediator in a family of SMAD proteins involved in TGFβ signaling. In this study, we investigated the molecular interactions between TGF-β1 and FLI-1 in AML cells and its functional role in TGF-β-mediated survival. In four AML cell lines, MV4;11, U937, NB4, and OCI-AML3, recombinant TGF-β1 (2ng/mL) induced the TGF-β downstream signaling targets plasminogen activator inhibitor-1 (PAI-1, mRNA) and/or Smad2 phosphorylation, which was reversed by anti-TGF-β neutralizing antibody 1D11 (Genzyme). No consistent change of Smad4 expression was observed in TGF-β1 treated cells. Treatment with rhTGF-β1 inhibited serum starvation-induced apoptosis in MV4;11, U937 and NB4, but not in OCI-AML3 cells. The anti-apoptotic effect of TGF-β1 was associated with G0/G1 cell cycle arrest, which was effectively reversed by anti-TGF-β antibody 1D11.

In MV4;11, U937 and NB4 cells, in which rhTGF-β1 promoted cell survival, rhTGF-β1 downregulated expression levels of FLI-1 mRNA and/or protein. However, FLI-1 was upregulated by rhTGF-β1 in OCI-AML3 cells. Since FLI-1 activation is known to cause cell proliferation associated with Ras pathway activation, we investigated MAPK signaling downstream of Ras. Changes in ERK phosphorylation levels after rhTGF-β1 treatment were fully concordant with FLI-1, whereby phospho-ERK was downregulated in MV4;11, U937, and NB4 cells, and upregulated in OCI-AML3 cells. These effects were reversed by anti-TGF-β antibody 1D11. In turn, rhTGF-β1 induced Matrix metalloproteinase-1 (MMP-1) mRNA which inversely correlated with FLI-1 expression. (U937; 6.9 fold increase, OCI-AML3; 3.1 fold decrease). It has been reported that ERK signaling upregulates MMP-1 expression, and that FLI-1 downregulates MMP-1 promoter activity in human fibroblasts. MMP-1, being responsible for degradation of collagenous proteins of ECM, correlates with poor prognosis in leukemia. We also observed that rhTGF-β1 induced significant upregulation of anti-apoptotic Bcl-2 in MV4;11, U937, and NB4 cells, but not in OCI-AML3 cells.

In summary, TGF-β-induced FLI-1 downregulation and ERK inactivation may be implicated in pathological matrix remodeling via oncogenic MMP-1 transcription in TGF-β abundant BM microenvironment. These findings suggest that FLI-1 and MMP-1 contribute to chemoresistance and poor outcomes in AML and represent potentially targetable molecular aberrations in AML.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.