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Sci Rep
2021 Mar 23;111:6685. doi: 10.1038/s41598-021-86021-8.
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Efficacy of combination treatment using YHO-1701, an orally active STAT3 inhibitor, with molecular-targeted agents on cancer cell lines.
Taniguchi K
,
Konishi H
,
Yoshinaga A
,
Tsugane M
,
Takahashi H
,
Nishisaka F
,
Shishido Y
,
Asai A
.
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Signal transducer and activator of transcription 3 (STAT3) plays a critical role in regulating cell growth, survival, and metastasis. STAT3 signaling is constitutively activated in various types of hematologic or solid malignancies. YHO-1701 has been developed as an orally available STAT3 inhibitor. Herein, YHO-1701 in combination with molecular-targeted agents was evaluated. Additive or synergistic effects were observed in a broad spectrum of "combination treatment + cell line" pairs. Of particular interest was the synergistic effect observed when YHO-1701 was combined with imatinib or dasatinib [breakpoint cluster region-abelson (BCR-ABL) inhibitors], osimertinib [epidermal growth factor receptor (EGFR) inhibitor], crizotinib, alectinib, or ceritinib [anaplastic lymphoma kinase (ALK) inhibitors]. The results further showed a close relationship between these synergistic effects and the cellular levels of the key molecules involved in the target pathways for YHO-1701 and each combination drug. The combination of YHO-1701 with alectinib resulted in significantly greater antitumor activity without exhibiting body weight loss in an NCI-H2228 [echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion] xenograft mouse model. Our results strongly suggest that the logical strategy in combination with the novel STAT3 inhibitor YHO-1701 and other mechanistically different targeted agents, could be a promising approach in future clinical settings.
Figure 1. Schematic representation of the mechanism of growth inhibition by YHO-1701 and other molecular-targeted drugs. ER2, human epidermal growth factor receptor 2; JAK, Janus kinase; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase. Blunt arrows show molecular-targeted agents inhibiting relevant pathways.
Figure 2. In vitro synergy of YHO-1701 in combination with already-available targeted agents for a total of 33 “combination + cell line” pairs. Drug interactions (synergism or antagonism) were analyzed using the combination index (CI) method. The antiproliferative effect of YHO-1701 and/or molecular-targeted agents was evaluated in a human cancer cell line panel using the WST-8 assay at 48 or 72 h of exposure. CI values were calculated and represented as a synergy “heat map” where a drug combination is synergistic (pink color) if CI ≤ 0.9; additive (white color) if CI is 0.9–1.1; and antagonistic (gray color) if CI > 1.1.
Figure 3. Synergism between YHO-1701 and imatinib in SUP-B15 cells. (A) The antiproliferative effect of YHO-1701 and/or imatinib was evaluated at 72 h of exposure. Dose–response curves are shown in the left panel. Combination index (CI) values are plotted as a function of fraction affected (Fa) in the right panel. Significance was determined with Tukey’s test. †††p < 0.001 versus the imatinib group. ‡p < 0.05; ‡‡p < 0.01; and ‡‡‡p < 0.001 versus the YHO-1701 group. (B) Phosphorylation/activation patterns of crucial molecules on BCR-ABL and STAT3 signaling pathways were analyzed by western blotting. SUP-B15 cells were left untreated or treated with YHO-1701 and/or imatinib at the indicated doses for 24 h. The blots were probed with primary antibodies specific for the target proteins. Images were cropped for clarity, and full-length blots are presented in Supplementary Fig. S3.
Figure 4. Synergistic antiproliferative interaction between YHO-1701 and osimertinib in H1975 cells. (A) The antiproliferative effect of YHO-1701 and/or osimertinib was examined at 72 h of exposure. Dose–response curves of YHO-1701 and/or osimertinib in H1975 cells (left panel) and their CI versus fraction affected (Fa) plots (right panel) are shown. Significance was determined with Tukey’s test. †††p < 0.001 versus the osimertinib group. ‡p < 0.05; ‡‡p < 0.01; and ‡‡‡p < 0.001 versus the YHO-1701 group. (B) Phosphorylation/activation patterns of relevant molecules on EGFR and STAT3 signaling pathways were assessed by western blotting. H1975 cells were left untreated or treated with YHO-1701 and/or osimertinib at the indicated doses for 24 h. The blots were probed with the respective primary antibodies. Images were cropped for clarity, and full-length blots are displayed in Supplementary Fig. S3.
Figure 5. Synergism between YHO-1701 and alectinib in H2228 cells. (A) The antiproliferative effect of YHO-1701 and/or alectinib was investigated at 72 h of exposure. Dose–response curves are represented in the left panel. Combination index (CI) values are plotted as a function of fraction affected (Fa) in the right panel. Significance was determined with Tukey’s test. ††p < 0.01; †††p < 0.001 versus the alectinib group. ‡p < 0.05; ‡‡p < 0.01; and ‡‡‡p < 0.001 versus the YHO-1701 group. (B) Phosphorylation/activation patterns of key molecules on ALK and STAT3 signaling pathways were examined by western blotting. H2228 cells were left untreated or treated with YHO-1701 and/or alectinib at the indicated doses for 24 h. The blots were probed with the respective primary antibodies. Cropped images are shown, and full-length blots are presented in Supplementary Fig. S3.
Figure 6. In vivo characterization of orally administered YHO-1701 when combined with alectinib at different levels of efficacy. Alectinib was used at 2 mg/kg/day (A–C) or 4 mg/kg/day (D–F). H2228 xenograft mice were randomized on day 1. Mice were treated with vehicle, YHO-1701, alectinib, or YHO-1701 + alectinib once daily for 4 weeks using a 5-day on, 2-day off schedule at indicated doses (n = 7). Changes in tumor volume (A,D), tumor weight on day 26 (B,E), and relative body weight (C,F). Significance was determined by Tukey’s test (B) or Steel–Dwass test (E). *p < 0.05; **p < 0.01; ***p < 0.001 versus the vehicle group. †p < 0.05; ††p < 0.01 versus the alectinib group. ‡‡p < 0.01 versus the YHO-1701 group. mpk, milligrams per kilogram of body weight; YHO, YHO-1701.
Figure 7. Downregulation of the downstream target survivin in tumor tissues. Lysates prepared from H2228 xenografts in Fig. 6E were subjected to further analyses. Survivin levels were determined by ELISA. Significance was determined by Tukey’s test. *p < 0.05 versus the vehicle group. mpk, milligrams per kilogram of body weight; YHO, YHO-1701.
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