Identification of Sox4 As Key Oncogene in Leukemias with Mutated or Silenced C/EBPα

Abstract 114

Transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) controls cell proliferation and myeloid differentiation. In 7∼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 molecular downstream events required for C/EBPA loss to trigger leukemogenesis are still unclear.

Based on microarray gene expression analysis, we here used a shRNA screening platform to search for mediators of leukemic outgrow of C/EBPα-deficient progenitor cells. In our screen, oncogene Sox4 was identified as a gene that was up-regulated in C/EBPα-deficient hematopoietic stem cells (HSCs, both lineage⁻c-kit⁺ScaI⁺ HSCs and SLAM⁺ HSCs) and whose down-regulation abrogated aberrant self-renewal ability and restored myeloid differentiation of C/EBPα-deficient stem/progenitor cells, 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 stem/progenitor cells 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 stem/progenitor cell 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 leukemia initiating cells of both leukemia models were enriched in immunophenotypically similar populations and exhibited comparable gene expression signatures. Similar to the C/EBPα knockout model, down-regulation of Sox4 by shRNA in LICs from murine C/EBPα mutant AML was also sufficient to abolish their augmented serial-replating ability. Importantly, 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.