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10780432ccr172773-sup-189704_2_supp_4595947_p4tgtd.pdf (2.45 MB)

Supplementary figures from Induction of Telomere Dysfunction Prolongs Disease Control of Therapy-Resistant Melanoma

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posted on 2023-03-31, 19:41 authored by Gao Zhang, Lawrence W. Wu, Ilgen Mender, Michal Barzily-Rokni, Marc R. Hammond, Omotayo Ope, Chaoran Cheng, Themistoklis Vasilopoulos, Sergio Randell, Norah Sadek, Aurelie Beroard, Min Xiao, Tian Tian, Jiufeng Tan, Umar Saeed, Eric Sugarman, Clemens Krepler, Patricia Brafford, Katrin Sproesser, Sengottuvelan Murugan, Rajasekharan Somasundaram, Bradley Garman, Bradley Wubbenhorst, Jonathan Woo, Xiangfan Yin, Qin Liu, Dennie T. Frederick, Benchun Miao, Wei Xu, Giorgos C. Karakousis, Xiaowei Xu, Lynn M. Schuchter, Tara C. Mitchell, Lawrence N. Kwong, Ravi K. Amaravadi, Yiling Lu, Genevieve M. Boland, Zhi Wei, Katherine Nathanson, Utz Herbig, Gordon B. Mills, Keith T. Flaherty, Meenhard Herlyn, Jerry W. Shay

Supplementary Fig. 1: Related to Fig. 2 (A) The percentage of apoptotic and dead cells indicated as PSVue 643+ cells in each of 12 BRAF-mutant melanoma cell lines treated with PLX4720 or 6-thio-dG at indicated doses for 120 hours. Cells were then harvested and co-stained with PSVue643 and Propidium iodide (PI). The average of 2 biological replicates was plotted. (B) SA-β-gal staining of 4 BRAF-mutant melanoma cell lines treated with 6-thio-dG at 5μM for 9 days. A representative image of 3 biological replicates was shown for each experimental sample. (C and D) Mouse weights of 1205Lu (C) and A375 (D) xenografts in each treatment group were shown. upplementary Fig. 2: Related to Fig. 4 (A) Long-term cell growth assay of 4 melanoma cell lines that acquired resistance to MAPKi treated with 6-thio-dG at indicated doses for 12 days. Cells were then fixed and stained with 3 crystal violet. A representative image of 2 biological replicates was shown for each experimental condition. (B-D) Tumor volumes of WM9 BR (B), UACC-903 BR (C) and A2058 CR (D) xenografts that were treated with the vehicle control and 6-thio-dG at indicated doses. (E) The ssGSEA plot of 11 MSigDB gene sets related to telomere and telomerase that were significantly altered in LOX-IMVI BR cells treated with BIBR 1532 (BIBR) or 6-thio-dG (6dG). (F-I) The heatmaps of RPPA data depicting 30 proteins that were most significantly down-regulated in four representative BR cell lines treated with 6-thio-dG, including A375 BR (F), WM3936-1 (G), 1205Lu BR (H) and WM1552C BR (I). (J) Western blotting of proteins that were downregulated in LOX-IMVI BR cells treated with 6-thio-dG (6dG). (K) The immnohistochemical analysis of AXL was performed in A375 tumors that were treated with the control, 6-thio-dG and Dabrafenib. Supplementary Fig. S3: Related to Fig. 5 (A-C) The heatmaps of two telomere transcriptional gene signatures, two melanoma-specific gene sets and two MAPK pathway-related gene sets in three datasets in which transcriptomes of paired pre- and post-treatment tumor biopsies derived from patients who progressed on MAPKi were profiled. 5 Supplementary Fig. S4: Related to Fig. 5 6 The heatmaps of two telomere transcriptional gene signatures, two melanoma-specific gene sets and two MAPK pathway-related gene sets in paired pre-, on- and post-treatment tumor biopsies derived from 16 patients who were treated with immunotherapies. Supplementary Fig. S5: Related to Fig. 6 (A-E) The heatmaps of RPPA data depicting 30 proteins that were most significantly downregulated in 5 short-term cultures or cell lines derived from immunotherapy-resistant tumors that were treated with 6-thio-dG, including 13-456-3-3 (A), 15-1761-1-2 (B), WM4265-1 (C), WM4265-2 (D) and G43 (E).

Funding

NIH

DOD

Dr. Miriam and Sheldon G. Adelson Medical Research Foundation

Melanoma Research Foundation

Wistar Institute

MDACC

UTSW

History

ARTICLE ABSTRACT

Purpose: Telomerase promoter mutations are highly prevalent in human tumors including melanoma. A subset of patients with metastatic melanoma often fail multiple therapies, and there is an unmet and urgent need to prolong disease control for those patients.Experimental Design: Numerous preclinical therapy-resistant models of human and mouse melanoma were used to test the efficacy of a telomerase-directed nucleoside, 6-thio-2′-deoxyguanosine (6-thio-dG). Integrated transcriptomics and proteomics approaches were used to identify genes and proteins that were significantly downregulated by 6-thio-dG.Results: We demonstrated the superior efficacy of 6-thio-dG both in vitro and in vivo that results in telomere dysfunction, leading to apoptosis and cell death in various preclinical models of therapy-resistant melanoma cells. 6-thio-dG concomitantly induces telomere dysfunction and inhibits the expression level of AXL.Conclusions: In summary, this study shows that indirectly targeting aberrant telomerase in melanoma cells with 6-thio-dG is a viable therapeutic approach in prolonging disease control and overcoming therapy resistance. Clin Cancer Res; 24(19); 4771–84. ©2018 AACR.See related commentary by Teh and Aplin, p. 4629

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