Targeting Mcl-1 enhances DNA replication stress sensitivity to cancer therapy
Guo Chen, … , Paul W. Doetsch, Xingming Deng
Guo Chen, … , Paul W. Doetsch, Xingming Deng
Published December 11, 2017
Citation Information: J Clin Invest. 2018;128(1):500-516. https://doi.org/10.1172/JCI92742.
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Research Article Cell biology

Targeting Mcl-1 enhances DNA replication stress sensitivity to cancer therapy

Abstract

DNA double-strand breaks (DSBs) are mainly repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ) pathways. Here, we showed that myeloid cell leukemia sequence 1 (Mcl-1) acts as a functional switch in selecting between HR and NHEJ pathways. Mcl-1 was cell cycle–regulated during HR, with its expression peaking in S/G2 phase. While endogenous Mcl-1 depletion reduced HR and enhanced NHEJ, Mcl-1 overexpression resulted in a net increase in HR over NHEJ. Mcl-1 directly interacted with the dimeric Ku protein complex via its Bcl-2 homology 1 and 3 (BH1 and BH3) domains, which are required for Mcl-1 to inhibit Ku-mediated NHEJ. Mcl-1 also promoted DNA resection mediated by the Mre11 complex and HR-dependent DSB repair. Using the Mcl-1 BH1 domain as a docking site, we identified a small molecule, MI-223, that directly bound to BH1 and blocked Mcl-1–stimulated HR DNA repair, leading to sensitization of cancer cells to hydroxyurea- or olaparib-induced DNA replication stress. Combined treatment with MI-223 and hydroxyurea or olaparib exhibited a strong synergy against lung cancer in vivo. This mechanism-driven combination of agents provides a highly attractive therapeutic strategy to improve lung cancer outcomes.

Authors

Guo Chen, Andrew T. Magis, Ke Xu, Dongkyoo Park, David S. Yu, Taofeek K. Owonikoko, Gabriel L. Sica, Sarah W. Satola, Suresh S. Ramalingam, Walter J. Curran, Paul W. Doetsch, Xingming Deng

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Figure 8

Discovery of small molecule MI-223 as a lead compound that specifically binds to Mcl-1, inhibits HR DNA repair, and sensitizes cancer cells to DNA replication agents.

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Discovery of small molecule MI-223 as a lead compound that specifically ...
(A) Schematic illustration of screening strategies used to identify the lead compound Mcl-1 inhibitor MI-223 and its chemical structure. (B) Structural modeling of MI-223 in the BH1 domain binding pocket of Mcl-1 protein. (C) The binding affinity of MI-223 with WT Mcl-1 or ΔBH1 Mcl-1 deletion mutant protein was examined by isothermal titration calorimetry assay. The binding constant (KD) value was determined by fitting of the titration curve to a 1-site binding mode. Data represent the mean ± SD, n = 3 per group. (D) Fluorescence polarization assay was performed to measure the inhibitory constant (KI) value using purified Mcl-1 protein, MI-223, and fluorescence-labeled PUMA BH3 peptide. Data represent the mean ± SD, n = 3 per group. (E) H1299 cells were treated with increasing concentrations of MI-223 for 24 hours, followed by co-IP using Mcl-1 antibody. (F) HR repair efficiency was measured in H1299 DR-GFP cells in the absence or presence of increasing concentrations of MI-223. Data represent the mean SD, n = 3 per group. *P < 0.05, **P < 0.01, by 2-tailed t test. (G) H1299 cells were treated with olaparib (Ola, 20 μM), MI-223 (4 μM), or the combination for 24 hours, followed by immunostaining with Rad51 antibody. Rad51 foci were quantified by counting of at least 100 cells from each sample. Data represent the mean ± SD, n = 3 per group. ***P < 0.001, by 2-tailed t test. Scale bar: 25 μm. (H) H1299 cells were treated with Ola (2 μM), MI-223 (2 μM), or the combination, followed by colony formation assay. Data represent the mean ± SD, n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001, by 2-tailed t test.