Beclin-1 Phosphorylation By BCR-ABL Is Crucial for CML Leukemogenesis By Suppression of Autophagy
Authors: Yu Chuanjiang , Gorantla Sivahari Prasad , Mueller Tony , Lippert Lena , Yue Zhenyu , Zeiser Robert , Huber Tobias , Duyster Justus , Illert Anna Lena 
Published: October 2019

The constitutively activated 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 insensitivity of CML stem cells to TKI treatment is discussed as one major reason for this.

Recent evidence accumulates, 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 "antagonize" TKI-induced cell death. However, the molecular mechanism of BCR-ABL and TKI induced autophagy as well as its role as tumor suppressor or promoter is poorly understood.

In our study, we aim to identify the precise role of autophagy and its´ effector molecules in a murine CML model. To test whether BCR-ABL regulates autophagy, we measured LC3 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. Consistent with this, Ba/F3 cells expressing BCR-ABL WT induce autophagy, whereas Ba/F3 cell expressing BCR-ABL-T315I fail to induce autophagy by nilotinib treatment, pointing to a BCR-ABL specific autophagy induction than an unspecific effect of TKI treatment. Next, we investigated the proteins involved in BCR-ABL mediated autophagosome formation. Recruitment of VPS34 and ATG14 to Beclin1 was increased in case of nilotinib treatment and could thereby positively regulate autophagosome formation, whereas Rubicon, a negative regulator was less recruited to the Beclin1-complex.

To further identify the impact of Beclin1 as a key regulator of autophagy in BCR-ABL-driven leukemia, we used a targeted genetic approach in a CML mouse model. Interestingly, mice transplanted with Belin1 knockdown, BCR-ABL expressing bone marrow showed a less aggressive disease with significantly lower WBC-count, leukemic burden and prolonged overall survival of the mice. In contrast, deletion of ATG5, another central regulator of autophagy, was not able to change disease onset or progression in the CML model.

To further clarify the function of Beclin1, we performed biochemical binding analyses and were able to show, that Beclin1 binds to BCR-ABL independent of BCR-ABL kinase activity and Beclin1 is phosphorylated by BCR-ABL. Interestingly, Beclin1 is an exclusive target of BCR-ABL and can not be phosphorylated by other aberrantly activated tyrosine kinases like Flt3-ITD, NPM-ALK and PDGFRA-D842V. In vitro kinase assay with active ABL-kinase confirm Beclin1 as a specific substrate of BCR-ABL. GST pulldown experiments mapped the N-terminal region of Beclin1 to interact with BCR-ABL. Cloning of different phospho-deficient mutants identified tyrosine residues Y233 and Y352 of Beclin1 as the crucial sites for specific BCR-ABL phosphorylation. To test the impact of BCR-ABL mediated Beclin1-phosphorylation on autophagy induction, we generated Beclin1 phospho-mimic (Y233E/Y352E) and phospho-deficient (Y233F/Y352F) mutations. Interestingly, nilotinib treatment fails to induce autophagy in cells expressing the Beclin1 phospho-mimic mutations, thereby highlighting the necessity of Beclin1 in BCR-ABL-mediated autophagy. Expression of Beclin1 mutations in Beclin1 knockout MEFs and K562 cells show decreased binding of UVRAG, ATG14 and VPS34 to Beclin1 Y233E/Y352E, suggesting an important role of Beclin1 phosphorylation for complex stabilization and autophagy suppression.

Taken together our findings identify Beclin1 as a specific substrate of BCR-ABL. Downregulation of Beclin1 is associated with a prolonged overall survival of BCR-ABL transplanted animals; direct phosphorylation of Beclin1 on Tyrosine residues Y233 and Y352 lead to LC3 inhibition and suppression of autophagy. Our results thereby highlight the importance of Beclin1 in BCR-ABL-mediated leukemogenesis and show, that autophagy induction in CML cells may be rather a specific Beclin1-BCR-ABL interaction effect than a general microenvironmental stress phenomenon.

Disclosures

No relevant conflicts of interest to declare.