THE GENE SIGNATURE IN C/EBPA DYSFUNCTIONAL AML PREDICTS

Background:

C/EBPa plays a crucial role in granulocytic development, and defects in this transcription factor have been reported in acute myeloid leukemia (AML). K562 cells stably transfected with an inducible C/EBPa-estrogen receptor fusion protein (C/EBPa-ER) have been used as a model for human granulocytic differentiation. When stimulated with b-estradiol (E2) to induce nuclear translocation of C/EBPa, these cells differentiate towards neutrophils.

 Aims:

The aims of this project are: 1. To identify genes upregulated upon C/EBPa activation, and therefore involved in granulocytic development. 2. To determine whether this set of genes, referred as the C/EBPa signature, is downregulated in AML patient samples. 3. To identify small compounds that could reactivate the C/EBPa signature, and promote granulocytic differentiation.

Results:

K562 C/EBPa-ER expressing cells were stimulated with 1 uM E2 or EtOH vehicle control, and gene expression profiles were determined by microarrays. By using prediction analysis of microarrays (PAM), we identified the C/EBPa signature, characterized by a set of 33 genes which are upregulated upon C/EBPa activation. We analyzed the expression of the C/EBPa signature in a cohort of 525 de novo AML patients, and identified a subset of 110 patient samples characterized by low expression of this signature. We referred to this group of patients as the C/EBPa dysfunctional subset. Remarkably, a large percentage of samples harboring C/EBPa biallelic mutations clustered inside this subset. We hypothesize that re-activation of the C/EBPa signature in the C/EBPa dysfunctional subset could have therapeutic potential. In search for small molecules able to reverse the low gene expression of the C/EBPa signature we applied the Connectivity Map. This analysis predicted a positive connectivity between the C/EBPa activation signature and histone deacetylase (HDAC) inhibitors. We showed that HDAC inhibitors (trichostatin A and vorinostat) reactivate expression of the C/EBPa signature in K562 cells. Next, we determined that patient samples with biallelic mutations in C/EBPa from inside the dysfunctional group cultured in the presence of HDAC inhibitors showed upregulation of cell surface granulocytic markers such as CD15 and CD11b. On the contrary, patient samples with biallelic mutations in C/EBPa, but clustering outside the dysfunctional group, had no significant changes in the same conditions. In addition, quantitative RT-PCR showed upregulation of granulocyte specific genes such as G-CSF-R (CSF3R), Gelatinase A (MMP2), C/EBPe (CEBPE), and lysozyme (LYZ) in HDAC inhibitor treated cells compared to vehicle control (EtOH) in samples from inside, but not from outside, the C/EBPa dysfunctional group.

Summary / Conclusion:

Altogether, our data identify HDAC inhibitors as potential candidates in the treatment of certain AMLs characterized by the downregulation of the C/EBPa signature.

Abstrakta v časopise Haematologica 2013, Suppl1

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