Identification of Genes Abnormally Expressed in Human MLL-AF4 Leukemia.

The t(4;11)(q21;q23) translocation is a hallmark of infant acute lymphoblastic leukemia (ALL), which results in the fusion of the MLL gene on chromosome 11 and the AF4 gene on chromosome 4. MLL-AF4 fusion is the most common consequence of chromosomal translocations in infant leukemia and is associated with a poor prognosis. To identify leukemia-related genes, we used the SAGE technique to compare gene expression profiles between two MLL-AF4 patient samples and one normal sample (CD19 + progenitor B cells; 216,464 tags in total). We identified 61 candidate genes that appear to be abnormally expressed in the leukemia samples (29 up- and 32 down-regulated). Remarkably, we found that many candidate genes appear to play important role in the development of B cells. In addition, many candidate genes can bind with and/or regulate other candidates in the candidate gene list. For example, SYK, BTK and BLNK can bind directly and regulate each other. SYK can also bind directly with TNFRSF1B. In addition, EBF may positively regulate BLK, while BLK can bind directly with BTK. All six of these genes are significantly down-regulated in MLL-AF4 leukemia samples. BTK, SYK and BLK are tyrosine kinases. BTK (B-cell progenitor tyrosine kinase) is a key regulator in B-lymphocyte differentiation and activation. BLK (B-lymphocyte-specific tyrosine kinase) is expressed only in B lymphocytes, and controls pre-B cell development. SYK (spleen tyrosine kinase) is widely expressed in hematopoietic cells, which can phosphorylate BLNK (B-cell linker protein). BLNK represents a central linker protein that bridges the B-cell receptor-associated kinases and may regulate B-cell function and development. EBF (early B-cell factor) is a tissue-specific and differentiation stage-specific DNA-binding protein, and mice lacking Ebf are unable develop B lymphoid cells. TNFRSF1B is strongly expressed on stimulated T and B lymphocytes. Moreover, previous studies indicate that BTK, BLK, SYK, BLNK and TNFRSF1B can positively regulate apoptosis, while BTK can also positively regulate differentiation. Thus, their down-regulation may inhibit apoptosis and differentiation, and thereby contribute to leukemogenesis. In contrast, GNA12, a transforming oncogene which can enhance proliferation and transformation and can bind directly with BTK, is significantly up-regulated in MLL-AF4 leukemia cells. Its up-regulation may also be important to leukemogenesis. Taken together, the deregulation of the important candidate genes may contribute to leukemogenesis through inhibiting apoptosis and differentiation while promoting proliferation of hematopoietic cells. We have validated the expression patterns of the candidate genes with real-time quantitative RT-PCR and are studying the functions and pathways of the validated candidate genes using RNAi and retrovirus transduction over-expression methods. In addition, we will also establish knock-in or knock-out mouse models for the most promising functional candidate genes to see the effect on the development of leukemia. Our studies will provide important insights into the complex functional pathways related to MLL rearrangements in the development of acute lymphoblastic leukemia, which may lead to more effective therapy for these leukemias.