Off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their antitumor activity in RAS-driven cancers
Yiming Miao, … , Frederick Nguele Meke, Zhong-Yin Zhang
Yiming Miao, … , Frederick Nguele Meke, Zhong-Yin Zhang
Published June 6, 2024
Citation Information: J Clin Invest. 2024;134(15):e177142. https://doi.org/10.1172/JCI177142.
View: Text | PDF
Research Article Oncology

Off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their antitumor activity in RAS-driven cancers

Abstract

Aberrant activation of RAS/MAPK signaling is common in cancer, and efforts to inhibit pathway components have yielded drugs with promising clinical activities. Unfortunately, treatment-provoked adaptive resistance mechanisms inevitably develop, limiting their therapeutic potential. As a central node essential for receptor tyrosine kinase–mediated RAS activation, SHP2 has emerged as an attractive cancer target. Consequently, many SHP2 allosteric inhibitors are now in clinical testing. Here we discovered a previously unrecognized off-target effect associated with SHP2 allosteric inhibitors. We found that these inhibitors accumulate in the lysosome and block autophagic flux in an SHP2-independent manner. We showed that off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their antitumor activity. We also demonstrated that SHP2 allosteric inhibitors harboring this off-target activity not only suppress oncogenic RAS signaling but also overcome drug resistance such as MAPK rebound and protective autophagy in response to RAS/MAPK pathway blockage. Finally, we exemplified a therapeutic framework that harnesses both the on- and off-target activities of SHP2 allosteric inhibitors for improved treatment of mutant RAS–driven and drug-resistant malignancies such as pancreatic and colorectal cancers.

Authors

Yiming Miao, Yunpeng Bai, Jinmin Miao, Allison A. Murray, Jianping Lin, Jiajun Dong, Zihan Qu, Ruo-Yu Zhang, Quyen D. Nguyen, Shaomeng Wang, Jingmei Yu, Frederick Nguele Meke, Zhong-Yin Zhang

×

Figure 2

SHP2-AIs repress autophagy independent of SHP2.

Options: View larger image (or click on image) Download as PowerPoint
SHP2-AIs repress autophagy independent of SHP2. (A) RFP-LAMP1–expressing...
(A) RFP-LAMP1–expressing U2OS cells were treated with DMSO, 10 μM SHP099, or IACS-13909 for 3 hours. Representative images of LAMP1 (red, lysosome marker), SHP099 (green and blue), or IACS-13909 (blue) and merged channels are displayed. Scale bars: 10 μm. (B) RFP-LAMP1–expressing WT or SHP2-KO HEK293 cells were treated with 10 μM SHP099 or IACS-13909 for 3 hours. Representative images of LAMP1 (red, lysosome marker), SHP099 (green and blue), or IACS-13909 (blue) and merged channels are displayed. Scale bars: 10 μm. (C) WT or SHP2–/– HEK293 cells were treated with a series of SHP099 or IACS-13909 concentrations for 6 hours. Total lysates were used for immunoblots. (D) A375 cells were treated with a series of SHP099 concentrations with or without the presence of 20 μM CQ for 6 hours. Total lysates were used for immunoblots. (E) U2OS cells were treated with 10 μM SHP099, TNO155, or IACS-13909 for 6 hours and visualized with transmission electronic microscopy. Scale bars: 1 μm. (F) HEK293 cells were treated with 10 μM SHP099, RMC-4550, or IACS-13909 for 6 hours with or without 20 μM CQ pretreatment for 3 hours. Total lysates were used for immunoblots. Lanes were run on the same gel but were noncontiguous. (G) EGFP-mCherry-LC3–expressing U2OS cells were treated with 0.5 or 5 μM IACS-13909 or DMSO for 6 hours. Representative images of EGFP (green, pH sensitive), mCherry (red, pH insensitive), and merged channels are displayed. Scale bars: 10 μm. Autophagic index indicates the ratio of the areas of mCherry+ puncta to EGFP+ puncta. Mean autophagic index is plotted, with each individual data point representing 1 analyzed cell field (5–10 fields total) from 3 independent experiments (labeled with different colors). Significance was determined by 1-way ANOVA followed by Dunnett’s multiple-comparison test. ***P < 0.001. (H) Autophagy inhibition levels were determined by quantification of LC3-II/I ratio changes in Supplemental Figure 1A. To determine the EC50, a 5-fold increase of LC3-II/I ratio in comparison with DMSO was defined as 100% autophagy inhibition. Representative data from 3 independent experiments displayed for all panels.