Phosphorylation of Beclin 1 by BCR-ABL suppresses autophagy in CML

Media type: E-Book

Title: Phosphorylation of Beclin 1 by BCR-ABL suppresses autophagy in CML

Contributor: Yu, Chuanjiang [Verfasser]; Duyster, Justus [Akademischer Betreuer]; Zeiser, Robert [Akademischer Betreuer]

Corporation: Universitätsklinikum Freiburg ; Albert-Ludwigs-Universität Freiburg, Fakultät für Biologie ; Albert-Ludwigs-Universität Freiburg, Fakultät für Biologie ; Albert-Ludwigs-Universität Freiburg

imprint: Freiburg: Universität, 2016

Extent: Online-Ressource

Language: English

DOI: 10.6094/UNIFR/11354

Identifier:

Keywords: Chronisch-myeloische Leukämie ; Autophagie ; (local)doctoralThesis

Origination:

University thesis: Dissertation, Albert-Ludwigs-Universität Freiburg, 2016

Footnote:

Description: Abstract: The constitutively active chimeric tyrosine kinase BCR-ABL is critical for initiation, progression and maintenance of chronic myelogenous leukemia (CML). Imatinib and second-generation BCR-ABL tyrosine kinase inhibitors (TKIs) serve now as standard therapies for Ph+ patients. However, disease persistence occurs frequently and one of the major reasons was considered to be insensitivity of CML stem cells to TKI treatment. Recently accumulated evidence argues that, autophagy, a genetically regulated process of adaptation to metabolic stress, is involved in TKI-induced cell death. It is hypothesized, that TKI-induced autophagy could allow CML stem cells to become metabolically dormant enabling their survival under conditions that may mimic growth factor deprivation and thereby "counter" TKI-induced cell death. However, the molecular mechanism of TKI-induced autophagy in BCR-ABL+ CML, as well as its role in malignant progression is poorly understood. It has been shown that autophagy functions in CML in vitro, however, les is known in vivo. This study aimed to identify the precise role of autophagy and its effector molecules in a murine CML model. To identify the impact of autophagy in BCR-ABL-driven leukemia, a targeted genetic approach to knockdown Beclin 1 as a key regulator of autophagy in a CML mouse model was used. Interestingly, mice transplanted BCR-ABL expressing bone marrow harboring Beclin 1 knockdown showed a less aggressive disease with significantly lower WBC-count, leukemic burden and prolonged overall survival of the mice compared to mice transplanted with BCR-ABL + Beclin 1 WT BM. To further test whether BCR-ABL regulates autophagy, LC3 was measured as a marker for autophagy in BCR-ABL+ K562 cell. Interestingly, inhibition of BCR-ABL activity by nilotinib led to increased LC3-II expression and punctual LC3 accumulation, indicating that BCR-ABL activity can suppress autophagy. Next, the proteins involved in BCR-ABL mediated autophagosome formation were investigated. Recruitment of VPS34, UVRAG and ATG14 to Beclin 1 was increased in case of nilotinib treatment and could thereby positively regulate autophagosome formation, whereas Rubicon, a negative regulator, was recruited less frequently to the Beclin 1 complex. To further clarify the function of Beclin 1, biochemical analyses were performed. It showed that Beclin 1 binds to BCR-ABL independent of BCR-ABL kinase activity and Beclin 1 is phosphorylated by BCR-ABL. To test the impact of BCR-ABL mediated Beclin 1 phosphorylation on autophagy induction, Beclin 1 phospho-mimic (Y233E/Y352E) and phospho-deficient (Y233F/Y352F) mutations were generated. Interestingly, nilotinib treatment failed to induce autophagy in cells expressing the Beclin 1 phospho-mimic mutation, thereby highlighting the necessity of Beclin 1 in BCR-ABL-mediated autophagy. Expression of Beclin 1 mutants in Beclin 1 knockout MEFs and K562 cells showed decreased binding of UVRAG, ATG14 and VPS34 to Beclin 1 Y233E/Y352E, suggesting an important role of Beclin 1 phosphorylation for complex stabilization and autophagy suppression. Interestingly, phospho-deficient Beclin 1 also delays BCR-ABL-driven leukemogenesis, demonstrating that Beclin 1 phosphorylation is crucial for BCR-ABL-mediated leukemogenesis. In contrast, deletion of Atg5, another central regulator of autophagy, did not impede disease onset or progression in the CML model. Taken together, these findings identify Beclin 1 as a specific substrate of BCR-ABL, thereby highlighting the importance of Beclin 1 in BCR-ABL-mediated leukemogenesis and showing that autophagy induction in CML cells may be rather a specific Beclin 1-BCR-ABL interaction effect than a general microenvironmental stress phenomenon

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Rights information: In Copyright

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