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C. Yu et al.
acquire BCL-ABL-independent resistance through autophagy inhibition by activation of mTOR.46 Therefore, we hypothesize that BECLIN-1 may play similar roles in the resistance-acquired signaling cascade. BECLIN-1 is a central autophagy mediating protein in mammalian cells, and EGFR and AKT kinase have been shown to phospho- rylate BECLIN-1 leading to inactivation of the protein and suppression of autophagy. Interestingly, active EGFR has been shown to phosphorylate the same tyrosine residues Y233/Y352 as BCR-ABL, further highlighting the impor- tance of these tyrosine residues for autophagy suppres- sion.42 In line with these previous findings, we have demonstrated in the present study that BCR-ABL phos- phorylates BECLIN-1 and thereby suppresses autophagy. Interestingly, this effect is mediated by the alteration of BECLIN-1 affinity to BECLIN-1 binding partners (ATG14, VPS34 and VPS15), known as the BECLIN-1 core complex. Our results therefore provide a novel explanation for the suppression of autophagy in CML and expand our knowl- edge regarding BECLIN-1-associated pathogenic mecha- nisms in BCR-ABL+ leukemia.
A role for BECLIN-1 has been proposed in various malignancies, such as breast or lung carcinomas. However, the precise role of BECLIN-1 in tumorigenesis remains unclear: On one hand, Beclin-1 is considered as a tumor suppressor and its overexpression is favorable for treat- ment of various solid tumors.51-54 Furthermore, low expres- sion of Beclin-1 is a marker of poor prognosis and enhanced aggressiveness in breast cancer55 and loss of one Beclin-1 allele leads to enhanced tumor development in mice. On the other hand, our present study demonstrates that knockdown of Beclin-1 prolongs the survival of BCR- ABL+ leukemic mice, which is consistent with a previous in vitro study in CML cell lines.16 Gene array analysis of CML patient samples revealed upregulated Beclin-1 levels in CML patients compared to healthy controls (fold change: 1.22; q-value: <0.1%).56 Importantly, we could show that CML patients exhibit a significant increase of phosphorylated BECLIN-1 levels. Targeting BECLIN-1 in specific approaches might thereby represent an elegant and alternative treatment option for TKI-resistant or intol- erant CML patients by rendering CML cells sensitive to targeted therapies.
Recently, a study uncovered a kinase-independent role of EGFR in autophagy, showing that inactive oncogenic EGFR reversely triggers autophagy.57 These findings sup- port the hypothesis that cells can develop TKI-resistance through autophagy induction, which might be caused or even triggered through the inactive oncogenic kinase itself. This sheds some light on the role of autophagy on cellular survival rather than cell death and gives a ration- ale to explore the combinatory effect of kinase inhibitors
with autophagy inhibitors. A number of drugs (Chloroquine, Bafilomycin A1, MAPK inhibitors and PI3K inhibitors) were described to have inhibitory effects on autophagy, however, most of them are poorly selec- tive, limiting their therapeutic application. Therefore, the development of highly specific and selective autophagy inhibitors remains a mandatory necessity for the success- ful evaluation of the therapeutic combination therapy with TKI in CML. Moreover, accurate target identifica- tion among major autophagy players is fundamental for successful therapeutic application: BECLIN-1 seems an attractive target, as its role in kinase-driven cancer is not only shown for solid cancers but also for hematopoietic malignancies in our study. For ATG5, we were not able to prove essential function in CML development in mice, whereas Liu et al. were able to demonstrate a crucial role of the protein in MLL-AF9 mediated AML induction in mice.61 ATG3 expression was shown to be indispensable for effective CML progression.14 These results indicate that autophagy induction is highly specific and stringent- ly regulated, not only depending on the particular malig- nancy, but also on the involvement of the master regula- tors.
Taken together, in this study we uncover a crucial role of BECLIN-1 in BCR-ABL mediated transformation in vivo and were able to identify a molecular mechanism by which BCR-ABL kinase activity regulates autophagy. We show that BCR-ABL binds and phosphorylates BECLIN-1 on tyrosine residues 233 and 352, thereby leading to alter- ations of the UVRAG-VPS15-ATG14-VPS34-RUBICON- BECLIN-1 complex. Moreover, the BCR-ABL/ BECLIN-1 interaction suppresses autophagy and thereby bypasses the negative effect of autophagy on cancer cell survival and proliferation. Importantly, these data may be of clini- cal relevance, as CML patients exhibit upregulated BECLIN-1 phosphorylation levels. Our findings provide a novel link between BCR-ABL and BECLIN-1 and shed some light on how specific oncogenes influence autophagy.
Funding
The authors would like to thank their funding agencies. This work was supported by research grants from the Deutsche José Carreras Leukämie-Stiftung (DJCLS R14/22 to JD and ALI and DJCLS 02 FN/2017 to TAM). ALI was supported by a research grant from the University Medical Center Freiburg and from the government Baden-Wur̈ttemberg (BSL). JD and TAM were supported by a DFG grant (FOR 2033). TBH was sup- ported by the DFG (CRC1140, CRC 992, HU 1016/8-1), by the BMBF (01GM1518C), by the Else-Kröner Fresenius Stiftung (NAKSYS), by the European Research Council-ERC grant 616891 and by the H2020-IMI2 consortium BEAt-DKD.
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