Abstract 5506: Inactivation of mTOR is a strategy for tumor resistance to ribosome biogenesis inhibition

Abstract Increased ribosome biogenesis supports the need for high translation capacity and is a hallmark of cancer. Targeting this process presents a promising strategy for cancer therapy. We have developed first-in-class inhibitors of RNA polymerase I (Pol I) transcription that block the first and rate-limiting step of ribosome biogenesis, rRNA synthesis. We find that Pol I inhibition by BMH-21 leads to a significant decrease in active ribosomes. We found a broad, but heterogenous efficacy in 18 cancer cell lineages. To identify genes that drive potential resistance to Pol I inhibition, we performed genome-wide CRISPR-Cas9 screens in human colorectal carcinoma cells. These positive selection screens identified high-confidence hits leading to drug resistance and included all key positive regulators of the mTORC1 complex. These findings are striking and counterintuitive given that mTOR is a major driver of ribosome biogenesis and cellular translational programs. The findings were validated using chemical inhibition of mTOR by Torin-1, a catalytic mTOR inhibitor and genetic knockout of an activator of mTOR signaling. Consistently, compromised mTOR activity led to resistance to BMH-21. However, loss of mTOR activity did not abrogate Pol I transcription inhibition by BMH-21. To assess the impact on protein translation, we used polysome profiling and found that BMH-21 treatment caused a severe defect in ribosome biogenesis. Surprisingly, mTOR inactivation partially rescued the translation repressed by Pol I inhibition, suggesting that this rescue could be pivotal for cell survival. To profile translational events, we performed Ribo-seq and RNA-seq in the BMH-21 sensitive and resistant cells. These multi-omics results revealed that mTOR inactivation decreased polysome-based translation on 5’TOP mRNAs encoding ribosomal proteins, suggesting that mTOR inactivation reduces ribosome occupation on ribosomal mRNAs. We propose that mTOR inactivation switches off polysome translation to rearrange active ribosomes for translation of survival-essential mRNAs. We further quantified newly synthesized proteins by AHA-labeling combined with TMT-mass spectrometry. These data show that distinct proteins continue to be translated when both Pol I transcription and mTOR activity are blocked. Next, we will examine preferentially translated proteins in resistant cells, and explore combination therapies to circumvent the drug resistance. In summary, we uncover a new non-genetic model of cancer drug resistance termed as translational fitness that enables cells to survive severe translational repression. These findings reveal an unexpected complication by mTOR inhibitory strategies and have implications for exploring effective drug combinations in cancer therapy. Citation Format: Wenjun Fan, Lijing Yang, Hester Liu, Steffie Pitts, Niladri Sinha, Rajeshkumar N.V., Hariharan Easwaran, Rachel Green, Marikki Laiho. Inactivation of mTOR is a strategy for tumor resistance to ribosome biogenesis inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5506.