Contributions
Abstract: S116
Type: Oral Presentation
Presentation during EHA22: On Friday, June 23, 2017 from 11:45 - 12:00
Location: Hall D
Background
Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with recurrent mutations that are of pathogenic and prognostic relevance. Mutations in FBXW7 are among the most common mutations in CLL, yet their functional consequences are unknown. FBXW7 is an E3 ubiquitin ligase that ubiquitylates oncoproteins like NOTCH1, HIF1-α and c-MYC and thereby targets them for proteasomal degradation.
Aims
Methods
FBXW7 mutations were analyzed via amplicon-based targeted next generation sequencing in primary CD19-sorted samples of previously untreated CLL patients (n=905) as well as in CLL (n=8), MCL (n=5), T-ALL (n=2), Burkitt lymphoma (n=1) and LCL cell lines (n=3). In silico modeling with PolyPhen-2 predicted a potential impact of the mutations on the structure and function of FBXW7. For functional analysis, FBXW7 mutations were induced using CRISPR/Cas9 in the CLL cell line HG3, which does not harbor a NOTCH1 mutation. Both in this CRISPR/Cas9 mutated cell line and in primary CLL cells with FBXW7 mutations, the protein levels of FBXW7 substrates were examined. In addition, we quantified NOTCH1 and HIF1-α activity with Luciferase reporter assay in FBXW7 mutated HG3 cell lines.
Results
Heterozygous mutations in FBXW7 were found in 41/905 (4.5%) of CLL patients. The most common mutations of FBXW7 were missense mutations (32/41) that target the substrate binding domain of the FBXW7 protein as well as non-sense mutations (4/41). Interestingly, 5 patients harbored two concurrent FBXW7 mutations. By the use of the PolyPhen-2 software, all except one missense mutation in FBXW7 were predicted to be most likely damaging. No mutations in FBXW7 were found in the CLL, MCL and LCL cell lines analyzed. To determine the functional consequence of FBXW7 mutations in CLL, we induced either a heterozygous or a homozygous truncation of FBXW7 in the CLL cell line HG3, resulting in the loss of the substrate binding site of the WD40 domain. The homozygous truncation of FBXW7 resulted in an increase of NOTCH1, HIF1-α and c-MYC protein levels, whereas no difference of Cyclin E protein amount was detectable. In addition, an elevation of NOTCH1 activity was found in both the heterozygously and homozygously truncated mutant cell lines in comparison to the wildtype HG3 cell line. To confirm this finding, protein levels of 5 CLL patients with FBXW7 mutations were analyzed with a similar outcome.
Conclusion
Session topic: 5. Chronic lymphocytic leukemia and related disorders - Biology
Keyword(s): Chronic Lymphocytic Leukemia, Ubiquitination, Notch, MYC
Abstract: S116
Type: Oral Presentation
Presentation during EHA22: On Friday, June 23, 2017 from 11:45 - 12:00
Location: Hall D
Background
Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with recurrent mutations that are of pathogenic and prognostic relevance. Mutations in FBXW7 are among the most common mutations in CLL, yet their functional consequences are unknown. FBXW7 is an E3 ubiquitin ligase that ubiquitylates oncoproteins like NOTCH1, HIF1-α and c-MYC and thereby targets them for proteasomal degradation.
Aims
Methods
FBXW7 mutations were analyzed via amplicon-based targeted next generation sequencing in primary CD19-sorted samples of previously untreated CLL patients (n=905) as well as in CLL (n=8), MCL (n=5), T-ALL (n=2), Burkitt lymphoma (n=1) and LCL cell lines (n=3). In silico modeling with PolyPhen-2 predicted a potential impact of the mutations on the structure and function of FBXW7. For functional analysis, FBXW7 mutations were induced using CRISPR/Cas9 in the CLL cell line HG3, which does not harbor a NOTCH1 mutation. Both in this CRISPR/Cas9 mutated cell line and in primary CLL cells with FBXW7 mutations, the protein levels of FBXW7 substrates were examined. In addition, we quantified NOTCH1 and HIF1-α activity with Luciferase reporter assay in FBXW7 mutated HG3 cell lines.
Results
Heterozygous mutations in FBXW7 were found in 41/905 (4.5%) of CLL patients. The most common mutations of FBXW7 were missense mutations (32/41) that target the substrate binding domain of the FBXW7 protein as well as non-sense mutations (4/41). Interestingly, 5 patients harbored two concurrent FBXW7 mutations. By the use of the PolyPhen-2 software, all except one missense mutation in FBXW7 were predicted to be most likely damaging. No mutations in FBXW7 were found in the CLL, MCL and LCL cell lines analyzed. To determine the functional consequence of FBXW7 mutations in CLL, we induced either a heterozygous or a homozygous truncation of FBXW7 in the CLL cell line HG3, resulting in the loss of the substrate binding site of the WD40 domain. The homozygous truncation of FBXW7 resulted in an increase of NOTCH1, HIF1-α and c-MYC protein levels, whereas no difference of Cyclin E protein amount was detectable. In addition, an elevation of NOTCH1 activity was found in both the heterozygously and homozygously truncated mutant cell lines in comparison to the wildtype HG3 cell line. To confirm this finding, protein levels of 5 CLL patients with FBXW7 mutations were analyzed with a similar outcome.
Conclusion
Session topic: 5. Chronic lymphocytic leukemia and related disorders - Biology
Keyword(s): Chronic Lymphocytic Leukemia, Ubiquitination, Notch, MYC