Yubo Liu

Bcl-2 phosphorylation confers resistance on chronic lymphocytic leukaemia cells to the BH3 mimetics ABT-737, ABT-263 and ABT-199 by impeding direct binding.

Although the ongoing clinical trials of ABT‐263 and ABT‐199 in chronic lymphocytic leukaemia (CLL) have indicated that BH3 mimetics hold considerable promise, understanding the mechanism of CLL resistance to BH3 mimetics remains a challenge.

Resistance to BH3 mimetic S1 in SCLC cells that up-regulate and phosphorylate Bcl-2 through ERK1/2.

B cell lymphoma 2 (Bcl‐2) is a central regulator of cell survival that is overexpressed in the majority of small‐cell lung cancers (SCLC) and contributes to both malignant transformation and therapeutic resistance. The purpose of this work was to study the key factors that determine the sensitivity of SCLC cells to Bcl‐2 homology domain‐3 (BH3) mimetic S1 and the mechanism underlying the resistance of BH3 mimetics.

A novel BH3 mimetic efficiently induces apoptosis in melanoma cells through direct binding to anti‐apoptotic Bcl‐2 family proteins, including phosphorylated Mcl‐1

The Bcl‐2 family modulates sensitivity to chemotherapy in many cancers, including melanoma, in which the RAS/BRAF/MEK/ERK pathway is constitutively activated. Mcl‐1, a major anti‐apoptotic protein in the Bcl‐2 family, is extensively expressed in melanoma and contributes to melanomas well‐documented chemoresistance. Here, we provide the first evidence that Mcl‐1 phosphorylation at T163 by ERK1/2 and JNK is associated with the resistance of melanoma cell lines to the existing BH3 mimetics gossypol, S1 and ABT‐737, and a novel anti‐apoptotic mechanism of phosphorylated Mcl‐1 (pMcl‐1) is revealed. pMcl‐1 antagonized the known BH3 mimetics by sequestering pro‐apoptotic proteins that were released from Bcl‐2/Mcl‐1. Furthermore, an anthraquinone BH3 mimetic, compound 6, was identified to be the first small molecule to that induces endogenous apoptosis in melanoma cells by directly binding Bcl‐2, Mcl‐1, and pMcl‐1 and disrupting the heterodimers of these proteins. Although compound 6 induced upregulation of the pro‐apoptotic protein Noxa, its apoptotic induction was independent of Noxa. These data reveal the promising therapeutic potential of targeting pMcl‐1 to treat melanoma. Compound 6 is therefore a potent drug that targets pMcl‐1 in melanoma.

Mechanism of synergy of BH3 mimetics and paclitaxel in chronic myeloid leukemia cells: Mcl-1 inhibition

Paclitaxel is an alternative chemotherapeutic agent for chronic myelogenous leukemia (CML) when primary or secondary resistance of tyrosine kinase inhibitors (TKI) is emerging, because paclitaxel could bypass the apoptotic deficiencies linked to p53 and fas ligand pathways in CML. However, high levels of Bcl-2 family proteins in CML could resist paclitaxel-induced apoptosis. Herein, we utilized two BH3 mimetics ABT-737 and S1 to study the potential of BH3 mimetics in combination with paclitaxel in treatment of CML cells and illustrated the mechanism by which BH3 mimetics synergize with paclitaxel. As a single agent, S1 could induce apoptosis in CML-derived cell line K562, whereas ABT-737 was largely ineffective. However, both of the two agents could efficiently synergize with paclitaxel through intrinsic apoptosis pathway. By using Bcl-2 siRNA, Bcl-XL siRNA or Mcl-1 siRNA, we found although each of the three members exhibited activities to block paclitaxel-induced apoptosis, Mcl-1 was the determinant for the synergistic effect between paclitaxel and ABT-737 or S1. Furthermore, paclitaxel/ABT737 synergized to drastically upregulate Bim to displace Bak from Mcl-1, whereas S1 directly binds Mcl-1 to release both Bim and Bak. As such, ABT-737 and S1 sensitized CML to paclitaxel by Mcl-1 inhibition, indirect inhibition through Bim antagonizing Mcl-1, or direct inhibition through binding to Mcl-1 itself. Finally, activation of JNK/Bim pathway was identified as the apical mechanism for ABT-737/paclitaxel synergism. Together, our results demonstrated potent synergy between BH3 mimetics and paclitaxel in the killing of CML cells and revealed an important role for Mcl-1 in mediating synergism by these agents.

Suppression of OGT by microRNA24 reduces FOXA1 stability and prevents breast cancer cells invasion

O-GlcNAc transferase (OGT) catalyzes the addition of O-GlcNAc to certain serine or threonine residue on a wide variety of cytosolic and nuclear proteins and regulates cellular activities such as signaling and transcription. Although there are emerging evidences that OGT plays important roles in breast cancer metastasis, the underlying mechanism is not fully understood. In this study, we demonstrated that up-regulation of OGT correlates with breast cancer cells invasion. Over-expression of OGT stimulates cells invasion, while OGT silence exhibits the opposite effects. OGT is further identified as a target of microRNA24 (miR24). miR24 down-regulates OGT expression and subsequently suppresses cells invasion. Re-expression of OGT significantly rescues miR24-mediated invasion repression. Furthermore, our data showed that FOXA1 is subjected to O-GlcNAcylation, which instabilizes FOXA1 protein and promotes breast cancer cells invasion. In conclusion, our results demonstrated that miR24 inhibits breast cancer cells invasion by targeting OGT and reducing FOXA1 stability. These results also indicated that OGT might be a potential target for the diagnosis and therapy of breast cancer metastasis.

Mechanism of Mcl-1 Conformational Regulation Upon Small Molecule Binding Revealed by Molecular Dynamic Simulation.

Inhibition of interactions between Mcl‐1 and proapoptotic proteins is considered to be a therapeutic strategy to induce apoptosis in cancer cells. Here, we adopted molecular dynamics simulation with molecular mechanics–Poisson Boltzmann/surface area method (MM‐PB/SA) to study the inhibition mechanism of three Mcl‐1 inhibitors, compounds 1, 2 and 3. Analysis of energy components shows that the better binding free energy of compound 3 than compounds 1 and 2 is attributable to the van der Waals energy (ΔEvdw) and non‐polar solvation energy (ΔGnp) upon binding. In addition to the excellent agreement with previous experimentally determined affinities, our simulation results further show a bend of helix 4 on Mcl‐1 upon compound 3 binding, which is driven by hydrophobic interaction with residue Val253, leading to a narrowed BH3‐binding groove to impede PumaBH3 binding. The computational result is consistent with our competitive isothermal titration calorimetry (ITC) assays, which shows that the competitive ability of compound 3 toward Mcl‐1/PumaBH3 complex is improved beyond its direct binding affinity toward Mcl‐1 itself, and compound 3 exhibits much more efficiency to compete with PumaBH3 than compound 2. Our study provides a new strategy to improve inhibitory activity on Mcl‐1 based on the conformational dynamic change.

S1 kills MCF-7/ADR cells more than MCF-7 cells: A protective mechanism of endoplasmic reticulum stress.

Drug resistance in chemotherapy for breast cancer is associated with high levels of P-glycoprotein (P-gp) as well as endoplasmic reticulum (ER) stress. In this paper, we aimed to evaluate the efficacy of a pan-BH3 mimetic S1 on drug-resistant MCF-7/ADR cells, and the roles of S1-induced ER stress in cell death. S1 exhibited greater and faster mitochondrial apoptosis in MCF-7/ADR cells than in MCF-7 cells. We demonstrated by Bax/Bak activation and cyrochrome c release that the p-glycprotein had little influence on S1 entering cells and hitting its targets in MCF-7/ADR cells. An IRE1-mediated ER stress response followed by c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) activation was specifically induced by S1 in MCF-7 cells, but not in MCF-7/ADR cells. Coimmunoprecipitation and western blotting analysis determined that ER stress played a protective role in S1-induced apoptosis by triggering Bcl-2 phosphorylation, which grabbed more pro-apoptotic proteins. The synergism effect of ERK inhibitor PD98059 with S1 confirmed the protective role of ER stress. Defective ER stress in MCF-7/ADR cells confers the more sensitivity toward S1.

Discovery of a small-molecule pBcl-2 inhibitor that overcomes pBcl-2-mediated resistance to apoptosis.

Although the role of Bcl‐2 phosphorylation is still under debate, it has been identified in a resistance mechanism to BH3 mimetics, for example ABT‐737 and S1. We identified an S1 analogue, S1‐16, as a small‐molecule inhibitor of pBcl‐2. S1‐16 efficiently kills EEE‐Bcl‐2 (a T69E, S70E, and S87E mutant mimicking phosphorylation)‐expressing HL‐60 cells and high endogenously expressing pBcl‐2 cells, by disrupting EEE‐Bcl‐2 or native pBcl‐2 interactions with Bax and Bak, followed by apoptosis. In vitro binding assays showed that S1‐16 binds to the BH3 binding groove of EEE‐Bcl‐2 (Kd=0.38 μM by ITC; IC50=0.16 μM by ELISA), as well as nonphosphorylated Bcl‐2 (npBcl‐2; Kd=0.38 μM; IC50=0.12 μM). However, ABT‐737 and S1 had much weaker affinities to EEE‐Bcl‐2 (IC50=1.43 and >10 μM, respectively), compared with npBcl‐2 (IC50=0.011 and 0.74 μM, respectively). The allosteric effect on BH3 binding groove by Bcl‐2 phosphorylation in the loop region was illustrated for the first time.

O-GlcNAc elevation through activation of the hexosamine biosynthetic pathway enhances cancer cell chemoresistance

Chemoresistance has become a major obstacle to the success of cancer therapy, but the mechanisms underlying chemoresistance are not yet fully understood. O-GlcNAcylation is a post-translational modification that is regulated by the hexosamine biosynthetic pathway (HBP) and has an important role in a wide range of cellular functions. Here we assessed the role of O-GlcNAcylation in chemoresistance and investigated the underlying cellular mechanisms. The results showed that the HBP has an important role in cancer cell chemoresistance by regulating O-GlcNAcylation. An increase in the levels of O-GlcNAcylation indicates an increased resistance of cancer cells to chemotherapy. Acute treatment with doxorubicin (DOX) or camptothecin (CPT) induced O-GlcNAcylation through HBP activation. In fact, the chemotherapy agents activated the AKT/X-box-binding protein 1 (XBP1) axis and then induced the HBP. Furthermore, the observed elevation of cellular O-GlcNAcylation led to activation of survival signalling pathways and chemoresistance in cancer cells. Finally, suppression of O-GlcNAcylation reduced the resistance of both established and primary cancer cells to chemotherapy. These results provide significant novel insights regarding the important role of the HBP and O-GlcNAcylation in regulating cancer chemoresistance. Thus, O-GlcNAc inhibition might offer a new strategy for improving the efficacy of chemotherapy.

Study of Binding Thermodynamics in the Optimization of BH3 Mimetics

The use of small molecule B‐cell lymphoma 2 homology domain 3 mimetics to neutralize the B‐cell lymphoma 2 protein is an attractive strategy for cancer treatment due to its ability to cause targeted cell apoptosis. We have previously reported the design and optimization of a series of B‐cell lymphoma 2 homology domain 3‐mimetics, called compounds 1–6. In this study, we evaluated the optimization of B‐cell lymphoma 2 homology domain 3‐mimetics from a thermodynamic perspective. Understanding the thermodynamic parameters of B‐cell lymphoma 2 homology domain 3‐mimetics plays a critical role in the development of B‐cell lymphoma 2 small‐molecule inhibitors. The thermodynamic parameters for the interactions of these compounds with the myeloid cell leukemia sequence 1 protein were obtained using isothermal titration calorimetry. Owing to compounds 1–6 overcoming enthalpy–entropy compensation, the affinities of them improved gradually. Toward binding to the myeloid cell leukemia sequence 1 protein, compound 6 was deemed optimal with an obtained Kd value of 238 nm, which is a 104‐fold improvement compared with 1. Analysis of the enthalpy and −TΔS efficiencies showed that ligand efficiencies with respect to molecular size are correlated with the enthalpic efficiencies. Notably, an enthalpy gain of 4.65 kcal/mol identified that an additional hydrogen bond is formed by 2 with myeloid cell leukemia sequence 1 compared with compound 1. For the first time, hydrogen bonding between a small‐molecule inhibitor of B‐cell lymphoma 2 was demonstrated experimentally.