Abstract 472: Interactions of the fusion protein Nup98-Hoxa9 with Pbx3, p300 and HDAC1: widening the targeted therapy window in acute myeloid leukemia (AML)
Authors: Rio-Machin Ana , Maiques-Diaz Alba , Rodriguez-Perales Sandra , Alvarez Sara , Salgado Rocio N. , Eguileor Álvaro , Torres Raul , Ramirez Juan C. , Cigudosa Juan C. 
Published: May 2015

The chromosomal translocation t(7;11)(p15,p15), that results in the oncogenic fusion protein Nup98-Hoxa9 (NH), appears in 1% of patients with AML and is associated with very poor prognosis and short overall survival. Despite the large severity of the leukemia induced by this fusion protein, the oncogenic events triggered by NH are poorly understood, although a potential role as an aberrant transcription factor has been proposed. We have generated a human Hematopoietic Progenitors (hHP) cellular model expressing NH constitutively to identify the molecular mechanisms supporting the malignancy of this fusion protein, facilitating the search for therapeutic targets.

We identified the DNA binding sites of NH by performing ChIP-seq experiments, which were validated by qRT-PCR analysis on ChIP selected DNA and Luciferase assays. Expression profiling was performed in hHP-NH and co-Immunoprecipitations (Co-IPs) were done to demonstrate the interaction of NH with different transcriptional regulators. Specific drug sensitivity of the hHP-NH model was assessed in cell proliferation assays.

Our work provides the first description of the DNA binding sites of NH, most of which are regulatory regions of genes involved in the development of AML. In particular, we demonstrate that NH induces the overexpression of MEIS1, HOXA9 and PBX3, transcription factors forming an activator complex that is a key element in the leukemic onset driven by other chromosome rearrangements. Interestingly, we show that NH directly interacts with this complex through Pbx3. To evaluate the biological relevance of the interaction of the MEIS1-HOXA9-PBX3 complex with NH, we have analyzed the sensitivity of hHP-NH to the HXR9 peptide (an inhibitor of the HOXA9-PBX3 interaction). Supporting our hypothesis, we observed an inhibitory effect on hHP-NH viability after HXR9 treatment. Finally, by combining the expression profile data from hHP-NH and the ChIP-seq results using GSEA analysis, we show that NH is able to induce both overexpression and down-regulation of its target genes. To provide evidences of the activator-repressor role of NH, we performed different Co-IPs that demonstrated its direct interaction with both p300 (transcriptional activator) and HDAC1 (transcriptional inhibitor).

Taken together, we show that the direct overexpression of the complex MEIS1-HOXA9-PBX3 is one of the pathogenic mechanisms induced by NH. As expected, the disruption of this complex with the HXR9 peptide in the hHP-NH model has a direct effect on cell viability. Furthermore, we show that NH interacts with this complex via PBX3 and also with p300 and HDAC1. The features and architecture of these interactions need to be further explored, but these findings allow us to consider the use of the HXR9 peptide or some HDAC inhibitors as possible treatments for these patients.

Citation Format: Ana Rio-Machin, Alba Maiques-Diaz, Sandra Rodriguez-Perales, Sara Alvarez, Rocio N. Salgado, Álvaro Eguileor, Raul Torres, Juan C. Ramirez, Juan C. Cigudosa. Interactions of the fusion protein Nup98-Hoxa9 with Pbx3, p300 and HDAC1: widening the targeted therapy window in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 472. doi:10.1158/1538-7445.AM2014-472