Abstract 3247: Functional CRISPR-Cas9 screens identify master regulators of resistance to chemical targeting of RNA polymerase I
Authors: Fan Wenjun , Liu Hester , Pitts Stephanie , Ford Brittany , NV Rajeshkumar , Laiho Marikki 
Published: June 2022

Increased ribosome biogenesis is a hallmark of cancer and targeting this process with RNA polymerase I (Pol I) inhibitors is a promising strategy for cancer therapy. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and induces degradation of the enzyme. This approach is effective across many cancer types. However, a heterogeneous response was observed in cancer cell lines emphasizing the need to increase knowledge of factors that underlie the response to this therapeutic strategy. To identify genes that modulate the response of cancer cells to BMH-21, we performed genome-wide CRISPR-Cas9-based positive selection screens in human colorectal carcinoma cells. These screens identified high-confidence hits accounting for BMH-21 drug resistance that included all key positive regulators of the mTORC1 complex. Given that p53 has been identified as a downstream effector of Pol I transcription stress, we conducted the screens in TP53 isogenic cells. Notably, the mTORC1 pathway hits were identified in all screens indicating that the resistance was p53 independent. These findings are particularly striking given that mTOR is a major driver of ribosome biogenesis and cellular translational programs. The findings were validated using chemical and genetic approaches. Torin-1, a catalytic mTOR inhibitor, was found to cause resistance to BMH-21. mTOR signaling pathway knockout and rescue cell lines were generated and tested for changes in the drug responses by growth, viability, colony formation and GFP competition assays. In each case, compromised mTOR activity led to resistance to BMH-21. However, compromised mTOR activity did not abrogate Pol I transcription inhibition by BMH-21. To assess impact on protein translation, we used polysome profiling. BMH-21 treatment was found to cause a severe ribosome biogenesis defect. Surprisingly, mTOR inactivation partially rescued the translation ability under the drug treatment suggesting that this translation is pivotal for cell survival. To uncover factors critical for the survival, we performed Ribo-seq and RNA-seq in the drug-treated BMH-21 sensitive and resistant cells. The profiling results revealed that mTOR inactivation led to elevated translation efficiency of mRNAs encoding ribosomal proteins. These findings suggest that mTOR inactivation evokes compensatory selective translation of ribosomal proteins under severe ribosome biogenesis defect caused by the Pol I inhibition. The findings indicate that mTOR inactivation regulates selective translation as means to bypass Pol I inhibition, and more generally, that maintenance of translational capacity strongly contributes to treatment resistance. These findings reveal an unexpected complication by mTOR inhibitory strategies as well as have implications on exploring drug combinations in cancer.

Citation Format: Wenjun Fan, Hester Liu, Stephanie Pitts, Brittany Ford, Rajeshkumar NV, Marikki Laiho. Functional CRISPR-Cas9 screens identify master regulators of resistance to chemical targeting of RNA polymerase I [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3247.