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10780432ccr202853-sup-247837_2_supp_6752784_qkgmhg.pptx (3.18 MB)

Supplementary Fig. S6 from Proteasome Inhibition Overcomes ALK-TKI Resistance in ALK-Rearranged/TP53-Mutant NSCLC via Noxa Expression

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posted on 2023-03-31, 22:20 authored by Azusa Tanimoto, Shingo Matsumoto, Shinji Takeuchi, Sachiko Arai, Koji Fukuda, Akihiro Nishiyama, Kiyotaka Yoh, Takaya Ikeda, Naoki Furuya, Kazumi Nishino, Yuichiro Ohe, Koichi Goto, Seiji Yano

Combination of alectinib and bortezomib induces apoptosis by upregulation of Noxa. A, Protein expression by western blotting in A925L cells treated with alectinib (3 µmol/L) and/or brotezomib (5 nmol/L) for 72 hours. B, the cells were treated with DMSO, alectinib (3 µmol/L), ixazomib (5 nmol/L), or a combination of alectinib (3 µmol/L) and ixazomib (5 nmol/L) for 9 d which were replenished every 72€‰h (right). The plates were stained with crystal violet and visually examined. A plate representative of three independent experiments is shown. C, Protein expression in A925L cells and H2228 cells transfected by siRNA control (scramble) or Noxa siRNA (si Noxa) which were treated with a combination of alectinib (3 µmol/L) and ixazomib (5 nmol/L) for 48 h. D, Western blotting of A925L cells overexpressing Noxa protein which were treated with alectinib (3€‰Âµmol/L) for 72 hours. E, Ubiquitinated Noxa protein in A925L cells which were treated with bortezomib (5€‰nmol/L) for 48 hours. N, C and B respectively correspond to non-specific, control and bortezomib. F, Protein expression in A925L cells detected by western blotting with immunoprecipitation of indicated proteins. Cell lines were treated with bortezomib (5€‰nmol/L) for 6 and 24 hours.

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ARTICLE ABSTRACT

In ALK-rearranged non–small cell lung cancer (NSCLC), impacts of concomitant genetic alterations on targeted therapies with ALK-tyrosine kinase inhibitors (ALK-TKI) are not yet well understood. Here, we investigated genetic alterations related to ALK-TKI resistance using clinico-genomic data and explored effective therapies to overcome the resistance in preclinical models through the identification of underlying molecular mechanisms. We used integrated clinical and next-generation sequencing data generated in a nationwide lung cancer genome screening project (LC-SCRUM-Japan). ALK-rearranged NSCLC cell lines expressing wild-type or mutant TP53 were used to evaluate cellular apoptosis induced by ALK-TKIs. In 90 patients with ALK-rearranged NSCLC who were treated with a selective ALK-TKI, alectinib, TP53 comutated patients showed significantly worse progression-free survival (PFS) than TP53 wild-type patients [median PFS, 11.7 months (95% confidence interval, CI, 6.3–not reached, NR) vs. NR (23.6–NR); P = 0.0008; HR, 0.33 (95% CI, 0.17–0.65)]. ALK-rearranged NSCLC cell lines that lost p53 function were resistant to alectinib-induced apoptosis, but a proteasome inhibitior, ixazomib, markedly induced apoptosis in the alectinib-treated cells by increasing the expression of a proapoptotic protein, Noxa, which bound to an antiapoptotic protein, Mcl-1. In subcutaneous tumor models, combination of ixazomib and alectinib prominently induced tumor regression and apoptosis even though the tumors were generated from ALK-rearranged NSCLC cells with nonfunctional p53. These clinical and preclinical results indicate concomitant TP53 mutations reduce the efficacy of alectinib for ALK-rearranged NSCLC and the combined use of a proteasome inhibitor with alectinib is a promising therapy for ALK-rearranged/TP53-mutated NSCLC.