C-Myb Is an Essential Downstream Target for Homeobox Mediated Transformation of Hematopoietic Cells.

Abstract

Overexpression of Hox genes is frequently associated with leukemogenic transformation of hematopoietic cells. In particular the abdominal-type HoxA genes together with their homeodomain containing dimerization partners of the TALE (three amino acid loop extension) family are well documented oncogenes in various hematologic malignancies. Paradigmatic examples are Hoxa9 and Meis1 that are coactivated by retroviral insertions in the BXH2 mouse leukemia model. In addition Hoxa9 is a fusion partner of NUP98 in acute myeloid leukemias with a t(7;11) and Meis1 as well as Hoxa9 are critical targets of MLL (mixed lineage leukemia) fusion proteins. Despite their important role in leukemogenesis little is known how they exert their transforming function. Here we used a novel system of transient transformation by an inducible oncoprotein to generate populations of primary hematopoietic cells that overexpress Hoxa9 in combination with Meis1. The RNA inventory of these cells was compared to control cells by Affymetrix array analysis. Interestingly the RNA of c-Myb (myeloblastosis leukemia virus oncogene) and of a known c-myb downstream target (Gstm1) was amongst the top ten genes upregulated by Hoxa9/Meis1. c-Myb overexpression was verified by Northern blot and quantitative RT-PCR. c-Myb levels also responded to the introduction of MLL-ENL, an oncoprotein that upregulates Hoxa9 and Meis1. Small interfering RNAs directed against c-Myb suppressed transformation by MLL-ENL but had no effect on transformation of hematopoietic precursors by E2A-HLF an oncogene that does not work by increasing Hoxa9/Meis1 levels. The anti c-Myb siRNA effect was abrogated by coexpression of a c-Myb derivative with a mutated siRNA target site. The coexpression of a dominant negative c-Myb mutant had a similar but weaker effect on MLL-ENL mediated transformation. Ectopic expression of c-Myb in hematopoietic precursor cells induced a mild differentiation block. However, c-Myb alone was not able to give a positive readout in a serial replating assay under conditions where the combined overexpression of Hoxa9 together with Meis1 was clearly transforming. In summary the results suggest that c-Myb is essential but not sufficient for the oncogenic activity of Hoxa9/Meis1.