Sox4 cooperates with Evi1 in AKXD-23 myeloid tumors via transactivation of proviral LTR

Myeloid leukemias in AKXD23 mice contain proviral insertions at Evi1, resulting in transcriptional activation. Although Evi1 is clearly involved in leukemia, gene transfer studies in mice with Evi1 fail to cause leukemia, arguing that cooperating events are necessary. We reanalyzed AKXD-23 tumors for cooperating proviral insertion and found that each tumor had a proviral insertion in Sox4, which encodes an HMG-box transcription factor. RNA analysis revealed these insertions cause increased Sox4 expression. Overexpression of Sox4 in 32Dcl3 cells markedly inhibited cytokine-induced granulocyte maturation, as documented by morphologic and mRNA analysis. Sox4-expressing cells had higher levels of transcripts associated with proliferation, including Evi1. Conversely, in leukemic cells that express Sox4 and bear provirally activated Evi1, suppression of Sox4 with short hairpin RNAs resulted in down-regulation of both Sox4 and Evi1. By cotransfection studies, Sox4 is able to transactivate the AKV long terminal repeat, which likely explains how Sox4 transcriptionally up-regulates provirally activated Evi1; however, Sox4 does not appear to regulate the native Evi1 promoter. We propose that Sox4 proviral activation is selected for in the setting of prior proviral activation of Evi1, because it transactivates the relatively weak LTR of AKV leading to higher Evi1 expression and consequent block to differentiation.