YL064 directly inhibits STAT3 activity to induce apoptosis of multiple myeloma cells

Abstract

Aberrant activation of signal transducer and activator of transcription 3 (STAT3) plays a critical role in the proliferation and survival of multiple myeloma. And inactivation of STAT3 is considered a promising strategy for the treatment of multiple myeloma. Here we show that the sinomenine derivative YL064 could selectively reduce the cell viability of multiple myeloma cell lines and primary multiple myeloma cells. Moreover, YL064 also induces cell death of myeloma cells in the presence of stromal cells. Western blot analysis showed that YL064 inhibited the constitutive activation and IL-6-induced activation of STAT3, reflected by the decreased phosphorylation of STAT3 on Tyr705. Consistent with this, YL064 inhibited the nuclear translocation of STAT3 and the expression of STAT3 target genes, such as cyclin D1 and Mcl-1. Using biotin- and FITC-labeled YL064, we found that YL064 could pull-down STAT3 from myeloma cells and colocalized with STAT3, suggesting that YL064 directly targets STAT3. Cellular thermal shift assay further demonstrated the engagement of YL064 to STAT3 in cells. Molecular docking studies indicated that YL064 may interact with STAT3 in its SH2 domain, thereby inhibiting the dimerization of STAT3. Finally, YL064 inhibited the growth of human myeloma xenograft in vivo. Taken together, this study demonstrated that YL064 may be a promising candidate compound for the treatment of multiple myeloma by directly targeting STAT3.

Fig. 1. YL064 selectively induces myeloma cell death.

a Chemical structure of YL064. b U266 and MM1.S cells were treated with the indicated concentrations of YL064 for 24 h and apoptotic cells were evaluated by Annexin V/PI double-staining assay. **p < 0.01, compared with control annexin V+/PI– cells, ^##p < 0.01, compared with control annexin V+/PI+ cells. c U266 and MM1.S cells were untreated or treated with indicated concentrations of YL064 for 24 h. The indicated proteins were detected by western blot. d Primary CD138⁺ MM cells were treated with YL064 (20 μM) for 24 h and CD138⁺ apoptotic cells were evaluated by Annexin V/PI double-staining assay. **p < 0.01, compared with control annexin V+/PI– cells, ^#p < 0.05, ^##p < 0.01, compared with control annexin V+/PI+ cells. e Primary PBMCs cells from healthy donors were treated with the indicated concentrations of YL064 (40 μM) for 24 h and cell viability was evaluated by Trypan blue exclusion assay

Fig. 2. YL064 inhibits constitutively active STAT3.

a, b U266 cells were treated with the indicated concentrations of YL064 for 24 h (a) or treated with YL064 (20 μM) for different times (b). Cells were collected and subjected to western blot analyses with specific antibodies against P-STAT3 (Tyr705), P-STAT3 (Ser727), and STAT3. c HeLa cells were transfected with p-STAT3-Luc and SV40-Renilla-Luc. Then, cells were treated with YL064 (20 μM) for 24 h, followed by stimulating with IL-6 for 20 min, and the luciferase activity were measured *p < 0.05, **p < 0.01. d U266 cells were treated with the indicated concentrations of YL064 for 6 h and STAT3 activity was examined by EMSA. e, f U266 cells were treated with YL064 (20 μM) for the indicated time points, and the mRNA level of cyclin D1, Mcl-1 were examined by RT-PCR and the indicated proteins were examined by western blot *p < 0.05, **p < 0.01

Fig. 3. YL064 inhibits IL-6-induced phosphorylation of STAT3.

a, b MM1.S cells were treated with the indicated concentrations of YL064 for 6 h (a) or YL064 at 20 μM for different times (b). The indicated proteins were detected by western blot analysis. c MM1.S cells were incubated with or without 20 μM YL064 for 6 h and then the intracellular distribution of STAT3 was analyzed by immunofluorescence

Fig. 4. YL064 inhibits HS-5 co-culture-induced activation of STAT3 in myeloma cells.

a HS-5, U266, and MM1.S cells were treated with the indicated concentrations of YL064 for 24 h. Cell viability was determined by Trypan blue assay. Values represented as graphs are the mean of three independent experiments with the standard deviation. b-d U266 and MM1.S cells were seeded on an established HS-5 stromal layer for 24 h, then the cells were treated with YL064 (20 μM) for additional 24 h. Subsequently, cells were separated from the HS-5 stromal layer, and apoptotic cells were examined by Annexin V/PI double-staining (b). **p < 0.01, compared with control annexin V+/PI– cells, ^##p < 0.01, compared with control annexin V+/PI+ cells. The indicated proteins were examined by western blot (c, d)

Fig. 5. YL064 interacts with STAT3 in cells.

a CETSA was performed on U266 cells as described in Materials and methods section. The thermal stability of STAT3 in cell lysate treated with YL064 (100 μM) at different temperatures (a) or different doses (b). The indicated proteins were evaluated by western blot. The intensity of the STAT3 bands was quantified by Quantity One software. Each experiment was repeated as least three times. c U266 cell lysate was incubated with biotin-YL064 (100 μM) or biotin for followed by pull-down with streptavidin-agarose beads. The precipitates were examined by western blot against STAT3 and STAT1. d U266 cells were treated with Fluorescein isothiocyanate (FITC) or 20 μM FITC-YL064 for 8 h, and then the colocalization of STAT3 (red) and YL064 (green) were examined by immunofluorescence staining. e The recombinant STAT3 protein was incubated with biotin-YL064 in the absence or presence of a ten-fold excess of unlabeled YL064 for 2 h, and the mixtures were blotted for biotin and STAT3

Fig. 6. Molecular docking of YL064 with STAT3.

a, b Predicted conformation of YL064 in the pocket of STAT3 SH2 domain (a). The molecular surface of STAT3 SH2 domain is shown in white, the phosphotyrosine peptide are colored yellow. YL064 and pTyr705 are shown in pink and yellow sticks, respectively. b Residues of STAT3 are shown in cyan sticks and labeled with residue names. The dashed lines in black represent hydrogen bonds. c U266 cells were treated with DMSO or YL064 (20 μM) for 24 h, the dimerization of STAT3 was examined in non-denaturating gel

Fig. 7. YL064 inhibits myeloma tumor growth in vivo.

a, b Mice bearing MM1.S MM tumors were treated with either YL064 (30 mg/kg; IP) or vehicle daily for 9 days. Tumor volume was measured every 2 days (a). Body weight were measured every 2 days (b). c The hematoxylin–eosin staining analysis of tumors. The indicated proteins were examined by immunohistochemistry (IHC) in tumor tissues (c). *p < 0.05, vehicle vs YL064-treated group. Scale bar = 50 μm