Abstract 2317: Identification of Sox4 as a key oncogene in leukemias with mutated or silenced C/EBPα.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) controls cell proliferation and myeloid differentiation. In ∼10% of patients with acute myeloid leukemia (AML) C/EBPA is either mutated or epigenetically silenced. C/EBPA mutated leukemias differ from C/EBPA silenced leukemias in prognosis and phenotype, yet both leukemias cluster together based on genome wide gene expression signatures, indicating a unifying mechanism of disease. So far, the key downstream events required for C/EBPA loss to trigger leukemogenesis are still unclear. Based on microarray gene expression analysis, we used a shRNA screening platform to search for mediators of leukemic outgrow of C/EBPα-deficient stem/progenitor cells (C/EBPα-/- HSC/HPCs, lineage-c-kit+ScaI+). In our screen, oncogene Sox4 was identified as a gene that was up-regulated in C/EBPα-/- HSC/HPCs and whose down-regulation abrogated aberrant self-renewal ability and restored myeloid differentiation of C/EBPα-/- HSC/HPCs, as demonstrated by in vitro serial-replating and differentiation assays. Chromatin immunoprecipitation confirmed the endogenous binding of C/EBPα at the proximal promoter of Sox4 in the HSC/HPCs enriched population (lineage-c-kit+) and the mature myeloid population (Mac1+Gr1+). In vitro promoter reporter assay demonstrated that wild-type human C/EBPA, but none of the C/EBPA mutants identified from AML patients, repressed Sox4 transcription through its binding to a highly conserved C/EBPα binding site. C/EBPα and Sox4 showed reciprocal expression patterns in both HSCs and various hematopoietic compartments during myeloid maturation of wild type mice. Furthermore, expression of Sox4 was up-regulated in HSCs of C/EBPα-deficient mice as well as in leukemia-initiating cells (LICs) of a murine C/EBPα mutant AML model. To further genetically dissect the role of Sox4 in driving leukemia in the absence of functional CEBPα, we generated Sox4, C/EBPα double deficient mice and observed that loss of Sox4 alleviated the abnormal HSC/HPCs expansion and defective myeloid programming caused by C/EBPα deficiency. In addition, comparisons of the murine C/EBPα mutant AML model with a Sox4-induced AML model revealed that LICs of both leukemia models were enriched in immunophenotypically similar populations and exhibited comparable gene expression signatures. Importantly, down-regulation of Sox4 by shRNA in LICs from murine C/EBPα mutant AML was sufficient to abolish their augmented serial-replating ability. Intriguingly, enhanced expression of SOX4 in AML patients with either mutated or epigenetically silenced C/EBPA compared to other AML subtypes confirmed the findings in our mouse model systems. Our data demonstrate that failure to suppress Sox4 expression is the underlying mechanism of leukemias with mutated or silenced C/EBPα. These data also uncover a promising rationale for a therapeutic target in these leukemias. Citation Format: Hong Zhang, Min Ye, Meritxell Alberich-Jorda, Giovanni Amabile, Henry Yang, Rob Welner, Philipp Staber, Veronique Lefebvre, Peter J.M. Valk, Ruud Delwel, Constanze Bonifer, Daniel G. Tenen. Identification of Sox4 as a key oncogene in leukemias with mutated or silenced C/EBPα. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2317. doi:10.1158/1538-7445.AM2013-2317