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. 2011 Dec 1;71(23):7312-22.
doi: 10.1158/0008-5472.CAN-11-1109. Epub 2011 Sep 21.

Src activation plays an important key role in lymphomagenesis induced by FGFR1 fusion kinases

Mingqiang Ren  1 , Haiyan Qin, Ruizhe Ren, Josephine Tidwell, John K Cowell
Affiliations

Src activation plays an important key role in lymphomagenesis induced by FGFR1 fusion kinases

Mingqiang Ren et al. Cancer Res. .

Abstract

Chromosomal translocations and activation of the fibroblast growth factor (FGF) receptor 1 (FGFR1) are a feature of stem cell leukemia-lymphoma syndrome (SCLL), an aggressive malignancy characterized by rapid transformation to acute myeloid leukemia and lymphoblastic lymphoma. It has been suggested that FGFR1 proteins lose their ability to recruit Src kinase, an important mediator of FGFR1 signaling, as a result of the translocations that delete the extended FGFR substrate-2 (FRS2) interacting domain that Src binds. In this study, we report evidence that refutes this hypothesis and reinforces the notion that Src is a critical mediator of signaling from the FGFR1 chimeric fusion genes generated by translocation in SCLL. Src was constitutively active in BaF3 cells expressing exogenous FGFR1 chimeric kinases cultured in vitro as well as in T-cell or B-cell lymphomas they induced in vivo. Residual components of the FRS2-binding site retained in chimeric kinases that were generated by translocation were sufficient to interact with FRS2 and activate Src. The Src kinase inhibitor dasatinib killed transformed BaF3 cells and other established murine leukemia cell lines expressing chimeric FGFR1 kinases, significantly extending the survival of mice with SCLL syndrome. Our results indicated that Src kinase is pathogenically activated in lymphomagenesis induced by FGFR1 fusion genes, implying that Src kinase inhibitors may offer a useful option to treatment of FGFR1-associated myeloproliferative/lymphoma disorders.

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Figures

Figure 1
Figure 1. Activation of Src is associated with FGFR1 chimeric kinese expression
A) Schematic outline of the FGFR1 break point junction in the chimeric FGFR1 fusion kinases shows conserved amino acids within the FRS2 consensus binding domain (amino acids 420-433) (21-22). (B) Cell proliferation rates in BaF3 cells transformed by ZMYM2-FGFR1 (ZMYM2-F), BCR-FGFR1 (BCR-F) or CEP110-FGFR1 (CEP110-F) show both IL-3 independence and an increased growth rate relative to cells carrying the empty vector (MIEGS). (C) Transduction of BaF3 cells (left panel) with the chimeric FGFR1 kinases identifies constitutive tyrosine phosphorylation in total cellular proteins using the anti-PY20 antibody compared with cells carrying the empty vector. These cells also show phospho-activation of Src kinase (right panel). (D) Ectopic expression of ZMYM2-FGFR1 in adherent 293T cells also leads to increased levels of phospho-Src. (E) Immunoprecipitation using anti-FRS2 antibodies co-precipitates the CEP110-FGFR1 fusion protein in CEP110-FGFR1 (CEP110-F) transformed BaF3 cells, but not in cells transduced with the empty vector. (F) When BaF3 cells expressing either ZMYM2-FGFR1 or CEP110-FGFR1 are treated with the TKI258 (400 nM) FGFR1 inhibitor, activation of both the FGFR1 fusion kinases and Src is inhibited. In contrast, TKI258 does not affect activation of either BCR-FGFR1 or Src in BCR-FGFR1 transformed cells.
Figure 2
Figure 2. Downregulation of activated Src correlates with growth inhibition
(A) Dasatinib treatment (100 nM) leads to suppression of Src kinase autophosphorylation in cells expressing ZMYM2-FGFR1, CEP110-FGFR1 and BCR-FGFR1. (B) Flow cytometric tracking of cell division with CellTrace™ Violet shows that 72 hours exposure to Dasatinib reduces cell proliferation in cells expressing ZMYM2-FGFR1 but not cells carrying the empty MIEG3 vector or treated with DMSO (vehicle).
Figure 3
Figure 3. Src is activated in primary lymphomas
Western blot analyses of phospho-activated and total Src levels in normal thymocytes (Thy) and lymph nodes (LN) from normal Balb/c mice compared with cells derived from lymph nodes (Lym) from mice with leukemia/lymphoma from the three different fusion kinase models. In these experiments Src activation is seen in the majority of primary tumors from the three different models.
Figure 4
Figure 4. Inhibition of Src activation results in apoptotic cell death
(A) Flow cytometric and western blot analysis of activated Src shows reduced phospho-Src levels in BaF3 cells stably expressing the three chimeric FGFR1 kinases following Dasatinib treatment. ZNF112 expresses ZMYM2-FGFR1, CEP2A and CEP5A cells express CEP110-FGFR1 and BBC1 expresses BCR-FGFR1. (B) Apoptosis and cell cycle analyses show that Dasatinib treatment (300 nM for ZNF112, CEP2A and CEP5A; 1000 nM for BBC, treated for 48h) remarkably increased cell apoptosis rate and decreased the percentage of cells in the S+G2 phase in ZMYM2-FGFR1 and CEP110-FGFR1 expressing cells. (C) Immunoprecipitation with anti-FRS2 in CEP2A and ZNF112 cells shows that both the phopho-FGFR1 fusion kinase and phospho-Src are present in the same immunocomplex. (D) Flow cytometry analysis shows infection with a dominant negative K295R/Y527F Src (pDNSrc) retroviral construct induces cell death in the cell lines expressing the chimeric FGFR1 kinases compared with the empty vector alone.
Figure 5
Figure 5. Knockdown of Src impairs transformation of 3T3 cells by ZMYM2-FGFR1
(A) Src-Yes-Fyn null mouse embryo fibroblasts (SYF−/−) and NIH3T3 (3T3) cells were infected with ZMYM2-FGFR1 or MIEG3 retroviral constructs. Stably infected pools were sorted for GFP positive cells and then analyzed by western blotting with anti-phospho-Src antibodies. The phospho-Src levels are increased in ZMYM2-FGFR1 transformed 3T3 cells. In contrast, total and phospho Src proteins are not detectable in triple knockout SYF−/− cells transduced with ZMYM2-FGFR1. B) Cell proliferation assays demonstrate that ZMYM2-FGFR1 expression increases cell proliferation in NIH3T3, but not in SYF−/− cells. C) Transduction with ZMYM2-FGFR1 significantly promotes anchorage-independent growth of 3T3 cells (3T3-FUS) but not SYF−/− cells (SYF−/−-FUS).
Figure 6
Figure 6. Dasatinib reduces lymphomogenesis of ZNF112 and CEP2A cells
(A) ZNF112 xenografted into Balb/c mice (n=10) and then treated with DMSO (vehicle) rapidly succumb. Xenografted mice treated with 20mg/kg (n=10) Dasatinib prolongs survival and at 40mg/kg (n=10) significantly prolongs survival. In these animals (B), spleen enlargement in the 40mg/kg group, are significantly reduced. Two weeks after treatment using both doses shows reduced numbers of GFP+ cells in the peripheral blood of Dasatinib treated mice (C). In the same way, (D) treatment of CEP2A xenografts (n=10) with 40mg/kg Dasatinib significantly prolongs survival and reduces spleen weight (E) in these animals. Reduced numbers of GFP+ cells is also seen in this model (F).
Figure 7
Figure 7. Constitutive Src activation in BCR-FGFR1 transduced human CD34+ and KG-1 cells
(A) Phospho-flow cytometry analysis showed that phospho-Src levels in BCR-FGFR1 transduced normal human CD34+ progenitor cells (GFP+BCR-FGFR1) is increased 62% compared with MIEG3 transduced CD34+ cells (GFP+MIEG3) based on the mean of florescent intensity (MFI) of the pSrc-PE channel. This experiment was repeated independently in triplicate and consistent results were obtained. (B) KG-1 cells, expressing FGFR1OP2-FGFR1, contain higher levels of phospho-Src compared with normal human mononuclear cells (hPBMN). Treatment with Dasatinib (100 nM), or FGFR1 inhibitors PD17073 (50 nM) or TKI258 (200 nM), inhibited Src activation in KG-1 cells. (C) Treatment with Dasatinib induces growth inhibition in KG-1 cells.
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