Rapid Analysis and Prediction of Drug Resistance in Leukemia By Novel Nascent RNA-Driven Flow Imaging Technologies and Biomarkers

Background:

Drug selectivity and resistance is a major obstacle to successful cancer therapy, including traditional chemotherapies (Vasan, N. et al. Nature 2019), immunotherapy (Hu-Lieskovan, S. et al. Future Oncol, 2021), and epigenetic therapy (Saliba, A.N. et al. Cancer Drug Resist,2021). However, no reliable technologies and biomarkers have been developed to analyze and predict drug resistance in clinical settings. Nascent RNAs composed of mostly non-coding RNAs undergo extensive modifications at co- and post-transcriptional levels. Nascent RNAs, and their associated modifications and modifying proteins (RMPs), such as RNA 5-methylcytosine (RNA:m ⁵C) and RNA cytosine methyltransferases (RCMTs), regulate almost all essential bioprocesses, including chromatin remodeling, DNA transcription, RNA processing/splicing and protein translation. Largely due to technology limitation, the role of nascent RNAs and RNA epigenetics remain largely unknown. Our previous publication first demonstrated distinct cell lineage-associated, RCMTs/BRD4-mediated, drug (azacitidine)-resistant transcriptionally active chromatin structures (TAC) at nascent (newly synthesized) RNAs in leukemia cells (Cheng, J.X. et al. Nat Commun. 2018). The goal of this study is to develop novel nascent RNA/TAC-driven technologies and biomarkers that enable us to rapidly analyze and predict drug resistance in clinical settings.

Results: 

1. Our experimental data demonstrated that TAC and RNA epigenetics regulate the resistance to venetoclax, a selective inhibitor of the anti-apoptotic protein BCL2, in leukemia cells. There is a significant, lineage-associated, increase in specific RCMTs, such as NSUN2 and NSUN1/NOP2, in venetoclax-resistant leukemia cells. Knockdown of NSUN2 and/or NSUN1 overcome venetoclax resistance in these leukemia cells.

2. Our data also demonstrated distinct patterns of drug- and lineage-specific RNA synthesis dynamics in drug-sensitive vs. -resistant leukemia cells. In drug-sensitive leukemia cells, dinaciclib, a potent a potent, selective small molecule inhibitor of CDKs inhibiting CDK1, CDK2, CDK5 and CDK9 at nano-molar concentrations (Parry, D. et al. Mol.Cancer Ther, 2010), completely inhibit nascent RNA synthesis within 3-5 min, while venetoclax and azacitidine partially inhibit nascent RNA synthesis within 15 min and 240 min, respectively. Such drug-induced inhibition of nascent RNA synthesis is completely independent of apoptosis and program cell death. In contrast, no drug-induced inhibition of nascent RNA synthesis is observed in drug-resistant leukemia cells.

3. Based on our data, we have developed a novel multifactorial system that targets the unique drug- and lineage-specific features of nascent RNA synthesis, TAC and RNA epigenetics for rapid analysis and prediction of drug resistance in clinical settings.

Conclusion:

Our data demonstrated distinct drug- and lineage-specific patterns of RNA synthesis in drug-sensitive vs. -resistant leukemia cells, which enabled us to develop novel nascent RNA/TAC-drive technologies and biomarkers for rapid analysis and prediction of anticancer drug resistance.