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Y. Lu et al.
end, cellular thermal shift assay (CETSA), a method used to evaluate the binding of compounds to target proteins in cells and tissue samples based on the biophysical principle of ligand-induced thermal stabilization of target pro- teins,43,44 was applied in NB4 cells. We observed that 2BP treatment markedly increased the thermal stability of RARα protein at temperatures examined compared to vehicle treatment (Figure 5A). Furthermore, RARα protein was accumulated by 2BP in a concentration-dependent manner (Figure 5B). Vinculin, a cytoskeletal protein associ- ated with cell-cell and cell-matrix junctions45 and pyruvate kinase M2(PKM2), a rate-limiting enzyme in glycolysis46,47 were taken as negative controls (Figure 5A-B). These data suggested that 2BP interacts with RARα in APL cells.
the modified peptide spectra (Figure 6B).
To verify the binding of 2BP with Cys105 and Cys174,
The binding between 2BP and RARα protein was fur- ther evaluated by surface plasmon resonance (SPR) assay using a biacore platform. The sensorgrams showed that 2BP rapidly associated with immobilized recombinant RARα protein at an equilibrium dissociation constant of 28.97 nM (Figure 5C). Moreover, the response signal dur- ing the dissociation phase did not return to the baseline level for 2BP, indicating that 2BP could not be completely eluted from RARα (Figure 5C). These data suggested that 2BP is covalently bound to RARα protein. Experiments with biotin-tagged 2BP (hereafter named biotin-2BP10) fur- ther supported that biotin-2BP could covalently bind with recombinant RARα protein, and this binding displayed a time-dependent saturation (Figure 5D).
Finally, we knocked down the endogenous RARα and re-expressed flag-tagged Cys105/Cys174 DM RARα into NB4 cells and evaluated the effects of 2BP. The results showed that 2BP enhanced the differentiation effect of ATRA in RARα-WT expressing but not RARα-DM expressing NB4 cells, indicating that 2BP presents a syner- gistic effect with ATRA through binding with Cys105/Cys174 (Figure 6E). Consistent with this, DM RARα could not be efficiently accumulated by 2BP in the presence of ATRA in NB4 cells (Figure 6F). Collectively, these data indicated that 2BP enhanced the ATRA-induced differentiation through binding to Cys105/Cys174 within RARα protein.
Mechanistically, covalent binding of 2BP with targets is more possible between the α-halo-carbonyl group and cysteines within proteins.10 Thereafter, we compared the effect of 2BP analogs, including palmitate acid (PA), 16BP and 12BP with 2BP on ATRA-induced differentiation of APL cells. These analogs either lack a bromine atom or have a bromine atom in a different position (Figure 5E). The results showed that, unlike 2BP, the three compounds did not present a synergistic effect with ATRA as evi- denced by the percentage of CD11b-positive cells (upper panel, Figure 5F). In addition, the three compounds did not accumulate RARα, as 2BP did (bottom panel, Figure 5F). These data indicated that the α-halo-carbonyl group is essential for the binding of 2BP with RARα.
Cys105 and Cys174 of RARα is the binding site for 2BP
To further determine the specific cysteine (Cys) residue that is modified by 2BP in RARα protein, we purified and incubated recombinant RARα protein with 2BP, followed by mass spectrometry (MS) analysis. There are 18 cys- teines located in different domains within RARα protein (Figure 6A). We identified 90% of the RARα protein sequence and 15 cysteine-containing peptides of the recombinant RARα incubated with and without 2BP (data not shown). The m/z ratio of the Cys105-containing pep- tide SSGYHYGVSACEGCK (Figure 6B) and Cys174-con- taining peptide KKEVPKPECSESY (Figure 6C) was meas- ured as 1,660.66 and 1,579.75 in the absence of 2BP and 1,857.86 and 1,776.96 in the presence of 2BP. The calculat- ed mass shift was consistent with the addition of one mol- ecule of 2BP. As for Cys105, MS/MS analysis of both unmodified and modified Cys105-containing peptide gave a partial series of y-ion fragments corresponding to the predicted sequence. Both MS/MS spectra had the same mass from y1 to y4, whereas the mass shifted 254.22 Da for the Cys105-containing fragment (from y5 to y12) in
Discussion
For the past decades, much effort has been devoted to identifying novel compounds with specific targets that would maximize the therapeutic effects of ATRA.28,29,48,49 For example, our group reported that pharicin B, a novel natural ent-kaurene diterpenoid derived from Isodon pharicus leaves, stabilizes RARα protein and presents syn- ergistic differentiation induction with ATRA in AML cells. It can also overcome retinoid resistance in two ATRA- resistant NB4 subclones.48 Wang et al. identified a novel synthetic small compound, named LG-362B, targeting PML-RARα and blocking ATRA resistance on cellular dif- ferentiation and transplantable murine models.28 More recently, Li et al. reported that pseudokinase Tribble 3 (TRIB3) promotes PML-RARα-driven APL by interacting with PML-RARα and disturbing the TRIB3/PML-RARα interaction through an α-helix peptide Pep2-S160 pro- duced significant anti-APL effects.49 All these studies sought to elucidate APL pathogenesis and find more ther- apeutic options for APL patients.
we incubated synthesized biotin-2BP with purified wild- type (WT) or Cys105/Cys174 double-mutated (DM) RARα proteins. Mutation of Cys105/Cys174 remarkably diminished the binding of RARα with 2BP in vitro (Figure 6D), indicating that 2BP covalently modified Cys105 and Cys174 of RARα.
In the present work, we have demonstrated that 2BP presents synergistic differentiation induction with ATRA in APL cell lines, primary APL blasts and in an APL murine model. Moreover, 2BP overcomes ATRA resistance both in vitro and in vivo, demonstrating therapeutic potential in APL. The cellular target of 2BP here, differently to previously reported CPT1 in FAO and PAT in protein palmitoylation, is RARα protein which triggers differentiation of leukemia cells through transcriptional mechanism. The binding of 2BP with RARα prevented the degradation of RARα pro- tein and sustained its transcriptional activity, leading to the differentiation-enhancing effect of 2BP. Recently, multiple investigations have indicated that RAR is a potential drug target for cancer and metabolic diseases.50 Thus, our data provides a new candidate to probe potential pathophysio- logical and therapeutic roles of RARα.
Notably, 2BP also stabilized PML-RARα, which was well-documented to block hematopoietic differentiation through interfering transcriptional activity of RARα, and the therapeutic effects of both ATRA and ATO relied on the degradation of this fusion protein.15,18,51 However,
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haematologica | 2019; 104(1)