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. 2016 Jun 14;113(24):6731-6.
doi: 10.1073/pnas.1601537113. Epub 2016 May 31.

Oncogenic mutations in the FBXW7 gene of adult T-cell leukemia patients

Chien-Hung Yeh  1 , Marcia Bellon  1 , Joanna Pancewicz-Wojtkiewicz  1 , Christophe Nicot  2
Affiliations

Oncogenic mutations in the FBXW7 gene of adult T-cell leukemia patients

Chien-Hung Yeh et al. Proc Natl Acad Sci U S A. .

Abstract

Human T-cell leukemia virus type 1 (HTLV-I) is associated with adult T-cell leukemia (ATL), an aggressive lymphoproliferative disease with a dismal prognosis. We have previously described the presence of Notch1 activating mutations and constitutive Notch1 signaling in patients with acute ATL. In this study, we report a high frequency of F-box and WD repeat domain containing 7 (FBXW7)/hCDC4 mutations within the WD40 substrate-binding domain in 8 of 32 acute ATL patients (25%). Functionally, ATL FBXW7 mutants lost their ability to interact with intracellular Notch (NICD), resulting in increased protein stability and constitutive Notch1 signaling. Consistent with the loss-of-function found in ATL patients, expression of WT FBXW7 in several patient-derived ATL lines demonstrated strong tumor-suppressor activity characterized by reduced proliferation of ATL cells. Remarkably, two FBXW7 mutants, D510E and D527G, demonstrated oncogenic activity when expressed in the presence of HTLV-I Tax, mutated p53 R276H, or c-Myc F138C found in human cancers. Transforming activity was further demonstrated by the ability of the FBXW7 D510E mutant to provide IL-2-independent growth of Tax-immortalized human T cells and increase the tumor formation in a xenograft mouse model of ATL. This study suggests that FBXW7, normally a tumor suppressor, can act as an oncogene when mutated and may play an important role in the pathogenesis of ATL.

Keywords: FBXW7; HTLV; Notch; leukemia; oncogene.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
FBXW7 is a tumor suppressor in ATL cells. The expression of FBXW7 in ATL-transformed cell lines and normal PBMCs (A) or healthy donors and freshly isolated ATL samples were evaluated by real-time RT-PCR. The arrow indicates the patients carrying the D510E mutation. (B) Cellular proliferation of FBXW7 transduced ATL lines were evaluated by cell count after reexpression of FBXW7. The data were from two independent experiments and shown as the average with SD. Western blot showed the expression of FBXW7. Actin served as a loading control. (C) The localizations of FBXW7 mutations found in primary ATL samples (Table 1) are highlighted in yellow on the FBXW7 protein 3D structure. (D) Transcriptional activities of NICD in cells expressing FBXW7 or FBXW7 mutants were measured by CSL-luciferase reporter assay. Luciferase activities were normalized to the CSL-luciferase reporter vector alone. The data are from two independent experiments and are shown as the average with SD. Western blot showed the expression of tagged-FBXW7 and mutants. (E) Dimer formation between FBXW7 and mutant W425R was analyzed in 293T cells transfected with Myc-tagged FBXW7 and Flag-tagged W425R. IP FBXW7 and Western blot W425R showed the dimerization. NICD expression in 293T cells transfected with NICD, FBXW7 WT and a dose increase of W425R were analyzed by Western blot (Lower). Dox, doxycycline.
Fig. 2.
Fig. 2.
FBXW7 mutants activate the Notch signaling pathway. (A and B) RT-PCR for expression of FBXW7 and Hes1 after 48 h of doxycycline (Dox) induction in MT1 stable lines. Results were repeated at least twice and fold-change was calculated compared with GAPDH expression. Western blots confirm the induction of FBXW7 (A) and reduction of endogenous nuclear NICD in WT-expressing cells but not mutants of FBXW7 (B). Actin and cyclin A served as loading control. (C and D) ChIP assays on FBXW7 WT and mutant following induction in stably transduced MT1 lines using RT-PCR. ChIP assays were prepared using anti-Notch and amplified with Hes-1 specific primers. (C) MT1 cells cultured with or without GSI (1 µM for 72 h) served as an assay control for loss of NICD-Val1744 and Hes1 binding. Fold-change was calculated as a percent of the initial input material. Sequencing of the amplified product verified Hes1 amplification.
Fig. 3.
Fig. 3.
FBXW7 mutants reduced binding and ubiquitin-mediated degradation of NICD. (A) FBXW7-mediated degradation of NICD was analyzed by Western blot (WB) and compared with control plasmid (pcDNA). FBXW7 WT and R505C were used as positive and negative controls, respectively. The NICD expression was quantified as relative to control normalized as 100% (WT 53%, R505C 115%, W406R 56%, T416A 61%, W425R 80%, L443F 100%, S462P 117%, H468R 111%, D510E 105%, and D527G 91%). (B) Expression of FBXW7 mutants increased the half-life of NICD. The effect of FBXW7 or mutants on the half-life of NICD was analyzed by Western blot after 100 μg/mL cycloheximide (CHX) treatment for 0, 2, 4, and 6 h. Western blot for transfected NICD, FBXW7, and actin are presented. (C) The interaction between NICD and FBXW7 was analyzed in 293T cells transfected with Myc-tagged NICD and Flag-tagged FBXW7 WT or mutants. Immunoprecipitated NICD and WB FBXW7 showed the interaction between NICD and FBXW7 WT. (D) Interaction between S-phase kinase-associated protein 1 (SKP1) and FBXW7 was analyzed in 293T cells transfected with HA-SKP1 and Flag-tagged FBXW7 WT or mutants. IP SKP1 and Western blot FBXW7 showed the interaction between SKP1 and FBXW7. (E) FBXW7-mediated NICD ubiquitination was analyzed in 293T cells transfected with FBXW7 WT (lane 2) or D510E (lane 3), NICD, and HA-Ub (K63 or K48). Cells were treated with MG132 for 6 h before harvest. Immunoprecipitated NICD and Western blot Ub showed the ubiquitination level of NICD.
Fig. 4.
Fig. 4.
FBXW7 mutants promote cellular transformation. (A) FBXW7 mutants increase Tax-transforming activity. Rat1 cell colony formation was evaluated after cells were transfected with the indicated plasmids and puromycin selection. The transformation efficiencies were normalized to Tax alone, which was set as 100%. The data were from two independent experiments and shown as the average. Increased number of transformed foci observed for D510E and D527G were statistically significant. (B) Photographs of transformed foci were shown (AMG EVOS XL core with 20× objective). (C) Western blot showed the expression of Tax and FBXW7. Actin served as a loading control. NICD expression in 293T cells transfected with NICD, FBXW7 WT, and a dose increase of D510E were analyzed by Western blot (Lower). (D) FBXW7 mutants increase p53 R276H and c-Myc F138C transforming activities analyzed as in A. The transformation efficiencies were normalized to p53 R276H and c-Myc F138C, respectively. The data were from two independent experiments and shown as the average with SD. (E) WT4 cells infected with lentivirus encoding FBXW7 WT or D510E were cultured in medium with or without IL-2 for 8 wk. Photographs of cell colonies are shown (AMG EVOS XL core with 20× objective). (F) FBXW7 WT and mutant-mediated substrate degradation were analyzed by Western blot after transient transfection. (G) Cellular proliferation of MT1 cells expressing FBXW7 WT and D510E were evaluated by cell count. The data were from two independent experiments and shown as the average with SD. (H) Degradation of endogenous substrates NICD, c-Myc, and cyclin E by FBXW7, W425R, S462P, and D510E mutant were analyzed by Western blot in ATL cells (MT1).
Fig. 5.
Fig. 5.
FBXW7 D510E increased the tumor formation in vivo. (A) MT1 FBXW7 (n = 5) or D510E (n = 5) TET-On cells were injected into the right or left flank of NOG mice. Pictures are representative of excised tumors from injected mice. Western blot for NICD (Val-1744) expression in tumor samples is shown. (B) In vivo tumor growth curves, plotted as the average tumor volume (mm3) [calculated as the (width2 × length)/2]. P values were calculated using a two-sided student’s t-test between the tumor volumes for FBXW7 vs. D510E. (C) Tumor weight (in grams) for FBXW7 and D510E tumors taken at the time of sacrifice. P values were calculated using a two-sided student’s t-test between the tumor weight for FBXW7 vs. D510E. The mean tumor weight was indicated with a bar and green square, with the SD indicated.
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