Identification and validation of putative NUP98/HHEX target genes by comparative gene expression profiling. (A) Gene expression signature of bone marrow progenitors cells 72 hours after retroviral transduction expressing NUP98/HHEX in unsupervised analysis using hierarchic clustering method. (B) Venn diagram analysis of numbers of genes found to be regulated by NUP98/HOXA9 (left), NUP98/HHEX (right), or both fusion genes (middle). (C) Quantitative RT-PCR (Q-PCR)–based validation of genes regulated by NUP98/HOXA9 and NUP98/HHEX in murine bone marrow cells 72 hours after transduction. (D) Expression of selected putative targets in NUP98/HHEX-expressing murine bone marrow cells after 10 days (▬) or 4 weeks () of culture in medium containing IL-3, IL-6, and SCF (Q-PCR analysis). (E) Expression of selected putative NUP98/HHEX target genes in leukemic blasts from primary (▬) and secondary () transplantations. Note the excessive levels of Flt3 mRNA expression in NUP98/HHEX blasts (right panel). (F) In vitro growth curve where bone marrow cells harvested from diseased NUP98/HHEX mouse were grown in 10% FBS and 50 ng/mL FL in the presence or absence of 10 μM Flt3 inhibitor for a period of 6 days. The insert shows a high level of expression of Flt3 mRNA in analyzed cells prior to the addition of Flt3 inhibitor. (G) Expression of HOXA5, HOXA9, FLT3, and PBX3 in blasts from a patient with t(10;11)(q23;p15) and NUP98/HHEX. Quantitative RT-PCR analysis normalized to the levels observed in cord blood mononuclear cells from 2 healthy donors. Bars represent the variance from 3 independent experiments.