Fig. 6

tnFGFR1 activates KIT and FLT3 in primary leukemia cells. Western blot analysis of KIT and FLT3 expression in leukemic cells from primary, tnFGFR1 transduced stem cells shows upregulation of both genes, compared to those cells transduced with BCR-FGFR1m (A). A similar correlation was seen in primary human AML cells that either express or do not express tnFGFR1 (B). (C) qRT-PCR detection of KIT, FLT3 and FGFR1 mRNA expression for human AML samples used for western blotting in (B) shows increased levels for all three genes in tnFGFR1 expressing cells. The preferential activation of KIT and FLT3 is further validated by flow cytometry analysis of primary leukemia cell from mouse models (D). Using two different shRNAs targeting tnFGFR1 (E, above) in primary tnFGFR1 transformed bone marrow cells, qPCR analysis (N = 3) shows extensive knockdown of tnFGFR1, Flt3, and Kit mRNA compared with cells transduced with a scrambled shRNA (SCR). Western blot analysis of the same cells (E, below) shows effective reduction in FLT3 and KIT protein levels in cells treated with either shRNA. ChIP analysis in primary tnFGFR1 transformed cells isolated from mouse bone marrow, using primers P1 from an upstream intergenic region, P2 within the Kit (F, left) or Flt3 (F, right) promoters and P3 within intronic regions of the genes, shows enrichment only for the P2 defined promoter regions (N = 3). ChIP was performed using antibodies that are specific to tnFGFR1 and also to the MYC-tag that was included in the transfection construct, both of which show the same result. ChIP using IgG alone was performed as a background control. Kaplan-Meier analysis of an AML cohort (N = 422, GSE37642) demonstrates a highly significant decrease in overall survival in patients with high expression levels of both FLT3 and GZMB compared with those showing low-level expression. No significant change in survival was found for different levels of expression of FGFR1 or KIT (G). ns = not significant. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001