Skip to main content
Fig. 3 | Molecular Cancer

Fig. 3

From: A truncated derivative of FGFR1 kinase cooperates with FLT3 and KIT to transform hematopoietic stem cells in syndromic and de novo AML

Fig. 3

tnFGFR1 is expressed widely in primary leukemia cells from mouse models and AML patients and is produced by GZMB cleavage of the full-length FGFR1. Western blot analysis of mice (N = 3) with primary transduced cells shows the full length kinase is expressed at high levels relative to the truncated tnFGFR1 in both BCR-FGFR1 (A) and ZMYM2-FGFR1 (B) murine leukemic cells. After serial transplantation of these cells from the primary mice through 6 cycles, the serially transplanted leukemia cells consistently demonstrate dominant expression of the tnFGFR1 derivative. In these cohorts there is increased expression of GzmB that is proportional to the increase in tnFGFR1 levels. Analysis of 29 primary AML bone marrow samples and 4 patient-derived xenograft (PDX) samples from immune compromised mice (C) demonstrates the presence of the full length FGFR1 kinase in specific cases, most of which also show the presence of the truncated kinase. In several other cases, there is predominant expression of the truncated kinase. For AML#10 and AML9276, the second transplant shows increased levels of FGFR1 proteins as seen in the syngeneic transplants, with AML9276 showing preferable enrichment of tnFGFR1. BaF3 cells transformed with the full length kinase (BCR-FGFR1) are used as molecular size controls. qRT-PCR analysis using primers targeting sequences in the proximal region of FGFR1 (P1), across the granzyme B cleavage site (P2) and within the tnFGFR1 derivative (P3) in a subset of nine of the most representative human AML samples (D) shows no significant difference in the relative copy number of different regions of FGFR1 mRNA between the tnFGFR1 exclusive samples and samples expressing both the full-length and truncated FGFR1 proteins. Sanger sequencing across the granzyme B cleavage site (E) from three AML that show high levels of tnFGFR1 protein show no alternative splicing around the GZMB site. qRT-PCR analysis of GZMB mRNA levels in the same samples (F) shows that higher levels of tnFGFR1 protein is correlated with high levels of GZMB expression. Mutation of the GZMB recognition site prevents tnFGFR1 production in BaF3 cells transformed with BCR-FGFR1m, compared with its parental BCR-FGFR1 transformed cells (G). Longer exposure times (L-expo) reveals tnFGFR1 in the cells transduced with BCR-FGFR1 but not BCR-FGFR1m. When mouse BBC2 and human KG1 SCLL cell lines are treated with GZMB Inhibitor II, quantitation of the protein levels normalized to actin levels (ACTB) shows the production of tnFGFR1 is decreased accordingly with increased inhibitor concentration (H). ns = not significant. *p ≤ 0.05

Back to article page