Bi-steric mTORC1 inhibitors induce apoptotic cell death in tumor models with hyperactivated mTORC1
Heng Du, Yu Chi Yang, Heng-Jia Liu, Min Yuan, John M. Asara, Kwok-Kin Wong, Elizabeth P. Henske, Mallika Singh, David J. Kwiatkowski
Heng Du, Yu Chi Yang, Heng-Jia Liu, Min Yuan, John M. Asara, Kwok-Kin Wong, Elizabeth P. Henske, Mallika Singh, David J. Kwiatkowski
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Research Article Oncology

Bi-steric mTORC1 inhibitors induce apoptotic cell death in tumor models with hyperactivated mTORC1

Abstract

The PI3K/AKT/mTOR pathway is commonly dysregulated in cancer. Rapalogs exhibit modest clinical benefit, likely owing to their lack of effects on 4EBP1. We hypothesized that bi-steric mTORC1-selective inhibitors would have greater potential for clinical benefit than rapalogs in tumors with mTORC1 dysfunction. We assessed this hypothesis in tumor models with high mTORC1 activity both in vitro and in vivo. Bi-steric inhibitors had strong growth inhibition, eliminated phosphorylated 4EBP1, and induced more apoptosis than rapamycin or MLN0128. Multiomics analysis showed extensive effects of the bi-steric inhibitors in comparison with rapamycin. De novo purine synthesis was selectively inhibited by bi-sterics through reduction in JUN and its downstream target PRPS1 and appeared to be the cause of apoptosis. Hence, bi-steric mTORC1-selective inhibitors are a therapeutic strategy to treat tumors driven by mTORC1 hyperactivation.

Authors

Heng Du, Yu Chi Yang, Heng-Jia Liu, Min Yuan, John M. Asara, Kwok-Kin Wong, Elizabeth P. Henske, Mallika Singh, David J. Kwiatkowski

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Figure 3

De novo purine synthesis is suppressed by RMC-6272 in an mTORC1/JUN/PRPS1-dependent manner.

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De novo purine synthesis is suppressed by RMC-6272 in an mTORC1/JUN/PRPS...
(A) Purine metabolites following RMC-6272 (3 nM, 24 hours) or rapamycin (3 nM, 24 hours) treatment of HCV29 TSC1-null cells. (B) mRNA levels (RNA-Seq) of de novo purine synthesis enzymes and related TFs in HCV29 TSC1-null cells treated for 24 hours. (C and D) Quantitative reverse transcriptase PCR assessment of PRPS1 (C) and PRPS2 (D) mRNA levels in HCV29 cells treated with rapamycin (10 nM), MLN0128 (10 nM), RMC-4627 (3 nM), or RMC-6272 (3 nM) for different times. (E–G) IC50 curves of HCV29 cells supplied with 100 nM of IMP (E), AMP (F), or GMP (G) and treated with rapamycin, MLN0128, RMC-4627, and RMC-6272. (H) Immunoblot analysis of PRPS1 KD in 705 and HCV29 cell lines. (I and J) Cell proliferation assay after knockdown of PRPS1 in 705 and HCV29 cells. (K) mRNA levels of Prps1 and Prps2 in Jun CRISPR KO 105K and 705 cells. (L) PRPS1 mRNA expression is decreased after JUN CRISPR KO in multiple cell lines. (M–O) Correlation between mRNA expression level of PRPS1 and the PI3K/AKT/mTOR pathway (M), tumor cell proliferation (N), and DNA replication (O) in TCGA data. (P) H3K27ac and JUN CUT&RUN data for the 621-101 cell line show open chromatin with JUN binding near the transcriptional start site of PRPS1. A and B: Dots are independent measurements, and lines are the median (n = 3). C–G, I, and J: Each dot and error bar represent mean ± SD (n = 3). One-way ANOVA was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.