Roles of Bax, Bcl-xL and Bcl-2 in TEL-FGFR3 Associated Oncogenic Property.

We previously reported that TEL-FGFR3 in a patient with peripheral T-cell lymphoma and AML conferred IL-3 independency to Ba/F3 cells and activates MAPKs, p38, PI3K, Stat3 and Stat5 through its constitutive tyrosine kinase (TK) activity in TEL-FGFR3 transfected Ba/F3 cells (TF-V5). Recent report suggests that FGFR3 activates Stat5 through recruitment of Pyk2 (proline-rich tyrosine kinase 2) to its juxta-membrane (JM) domain. To gain insight into the roles of Pyk2-Stat5-Bcl-xL axis in TEL-FGFR3 associate tumorigenesis, we performed a functional analysis of a deletion mutant of TEL-FGFR3 which lacks the JM domain. Stable polyclonal transfectants of Ba/F3 cells expressing the deletion mutant of TEL-FGFR3(ΔJM-V5) grew in absence of IL-3 without spontaneous apoptosis by constitutive TK activity. ΔJM TEL-FGFR3 did not recruit Pyk2 and induced little phosphorylation of Stat3 and Stat5 compared to those of TF-V5, while it induced constitutive phosphorylation of MAPKs and PLCγ. In ΔJM-V5, expressions of Bcl-xL and Bcl-2 were abrogated to the level of Mock without IL-3. To elucidate a mechanism of IL-3 independent cell growth of ΔJM-V5 which does not express Bcl-xL and Bcl-2, we examined expression of pro-apoptotic Bcl-2 families and revealed that Bax was not expressed in ΔJM-V5. In TF-V5, Bax was constitutively expressed and FGFR3 TK inhibitor SU5402 inhibited Bax expression after 24hr. These results indicate that there might be a pathway from JM domain to regulate Bax expression. To examine the role of Bcl-xL and Bcl-2 in DNA damage-induced apoptosis, TF-V5 and ΔJM-V5 were X-irradiated with 5, 15, 30 or 50Gy respectively. TF-V5 had reduced susceptibility to apoptosis following X-irradiation when compared with Mock transfected cells without IL-3. In TF-V5 the presence of IL-3 did not affect susceptibility to X-ray-induced apoptosis at any dose levels. In contrast, when ΔJM was treated with IL-3, significant reduced susceptibility to X-ray irradiation at more than 15Gy was observed (apoptotic fraction following 30Gy irradiation after 24hr: 40% without IL-3; 20% with IL-3). In ΔJM cells, IL-3 induced over-expression of Bcl-xL and Bcl-2 and cell cycle arrested at G1 was observed following more than 15Gy X-irradiation. Taken together, it is supposed that Bcl-xL and Bcl-2 play an anti-apoptotic role only when Bax is induced by activated p53 in ΔJM-V5. Further, to investigate leukemogenesis of ΔJM-V5 in vivo, ΔJM-V5 cells were injected intravenously into BALB/c mice. As a result, although all 10 mice intravenously injected ΔJM-V5 developed a disease resembling leukemia with enlargement of spleen and died within 30 days after injection, they exhibited a significantly prolonged disease latency (median survival=23.6 days) compared to all 10 mice intravenously injected TF-V5 died of a similar disease (median survival=18.0 days). Our results indicate that ΔJM does not activate Stat3 and Stat5 without Pyk2 recruitment and not express Bcl-xL, Bcl-2 and Bax.

Conclusion: Both Bcl-xL and Bcl-2 are dispensable for a TEL-FGFR3 transforming property especially in ΔJM-V5 which does not express Bax. However, Bcl-xL and Bcl-2 might be necessary for full oncogenic property of ΔJM TEL-FGFR3.