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2-Bromopalmitate promotes APL differentiation
forced expression of PML-RARα could increase ATRA sensitivity in U937 cells52 and restore ATRA sensitivity in NB4.007/6 ATRA-resistant cells,53 demonstrating a dual role for the fusion protein in leukemogenesis. On the other hand, a number of compounds capable of restrain- ing ATRA-dependent PML-RARα proteolysis have been shown to enhance ATRA-induced differentiation,54-57 indi- cating that a mechanism independent of PML-RARα degradation that drives granulocytic maturation does exist. Therefore, how stabilized PML-RARα protein con- tributes to 2BP-enhanced cell differentiation deserves fur- ther exploration.
Combination of ATO with ATRA serves as a frontline treatment of APL. ATO is also used as the best salvage therapy agent for ATRA-resistant APL patients.15,19,20,31 Mechanistically, ATO induced PML-RARα degradation through direct binding to cysteine residues in PML moiety of the fusion protein.51 However, resistance to ATO in APL was reported by several groups.24-26 Here, we observed an increase of APL cell differentiation and leukemia mice sur- vival upon cotreatment of ATO with 2BP, suggesting that an approach combining ATO with 2BP warrants further investigation as a therapeutic strategy for APL patients.
The effects of 2BP on cell differentiation have been observed in neural stem cell and osteoblast.11-13 However, no direct differentiation-associated target of 2BP has been identified. Interestingly, different from our results, research from Chen et al. showed that 2BP treatment impaired ATRA-induced neuronal differentiation in vitro which involved the palmitoylation of P300 and acetyla- tion of histones H3 and H4.13 Further investigation of 2BP- RARα interaction in the context of neural cells may offer some clues for better understanding the controversial effect of 2BP towards ATRA.
Most of the 2BP-targeted enzymes, whether in lipid or nonlipid processing, contain cysteine residues in or near the enzyme active site, suggesting α-halo-carbonyl elec- trophilic alkylation mediates the observed irreversible inhibition.10 In the present work, we identified that of all the 18 cysteines within RARα sequence, Cys105 and
Cys174 are the major residues for 2BP binding. Substitution of WT RARα with Cys105/Cys174 DM sig- nificantly decreased the synergistic differentiation activity of 2BP as well as the accumulation of RARα protein in the presence of ATRA, indicating that despite the potential promiscuous cellular targets of 2BP,10 the binding of 2BP with RARα at Cys105/Cys174 is responsible for, albeit partially, preventing ATRA-triggered degradation of RARα which helps differentiation. Previous studies have showed that ATRA-triggered degradation of RARα was mediated by the ubiquitin-proteasome pathway.34 Our data thus suggested that the binding of 2BP may influence the pro- teasomal degradation of RARα protein.
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Overall, RARα modulation in the treatment of APL has generated considerable interest in the development of RAR modulators and uncovered a promising strategy for these diseases. Our discoveries demonstrate that by tar- geting RARα, 2BP not only helps ATRA-induced APL dif- ferentiation, but also reverts ATRA resistance in vitro and in vivo. Combining ATRA with 2BP could be a potential candidate for ameliorating ATRA-associated adverse effects and clinical relapsed APL.
Acknowledgments
The authors would like to thank Dr. Ai-Wu Zhou for his kind- ness in providing us the psumo3 vector. We appreciate Dr. Wei- wei Wang for his professional suggestions on identification of the binding site of 2BP.
Funding
This work was supported by National Natural Science Foundation (81370652, 81770146, 81721004, 81430061,81570124,31570824), National Basic Research Program of China (2015CB910403), Foundation for the author of National Excellent Doctoral Dissertation of China (201074), Grants from Science and Technology Committee of Shanghai (13431900501) and the Reformation Project in the Key Clinical Departments of Provincial Hospitals on Construction of Diagnosis and Treatment Capacity in Liaoning Province (LNCCC-A02-2015).
Chase JF, Tubbs PK. Specific inhibition of mitochondrial fatty acid oxidation by 2- bromopalmitate and its coenzyme A and
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