Background: The human genome comprises ~99% non-coding DNA sequences (ncDNAs), most of which are actively transcribed into various forms of non-coding RNAs (ncRNAs) that undergo extensive chemical modifications to regulate various functions of the subcellular organelles in human cells (Statello, et al.Nat Rev Mol Cell Biol 2020). However, the clinical potential of RNA modifications and RNA modifying proteins, i.e., RNA epigenetics, is largely unexplored. In a recent study (Cheng et al. Nat Commun 2018), we first demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 and NSUN2, directly interact with elongating RNA polymerase II (eRNAPII) and the bromodomain-containing protein 4 (BRD4) to form distinct active chromatin structures (ACS) that regulate drug resistance in leukemia cells. This proof-of-concept study enabled us to develop the first-generation of RNA epigenetics-driven drug-predictive system. The primary goal of this study is to validate and optimize this novel drug-predictive system with clinical myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Methods: We developed new technologies, including 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). Immunohistochemistry (IHC), western blot (WB), co-immunoprecipitation (co-IP), and dot blot were also used in this study. Results: Our data demonstrated significantly increased expression of the NSUN1, NSUN2 and other key components of the drug-resistant ACS in MDS/AML, which is correlated with disease progression and drug resistance in MDS and AML. We have developed novel technologies, i.e., EC- and PL-RCA-coupled CM and FCM (EC-/PL-RCA/CM & FCM) to visualize and quantify RCMTs-mediated ACS and nascent RNA synthesis in MDS/AML. We discovered nascent RNA synthesis as a highly sensitive and super-fast drug-predictive marker that is independent of cell death and apoptosis. Our data demonstrated that PL-RCA/FCM and EC-/CM have a very high sensitivity to detect the NSUN1/2-mediated ACS in leukemia cell lines and clinical MDS/AML specimens. Our preliminary data demonstrated that increased levels of NSUN1/2-mediated ACS detected by PL-RCA/FCM in pre-treatment MDS/AML can rapidly predict the responses to venetoclax and/or azacitidine. We are actively pursuing RNA-seq targeting NSUN1/2-associated nascent RNAs and ACS, which provides a novel strategy to dissect the functional genomics and RNA epigenetics underlying cancer drug resistance. Conclusions: We have developed a novel RCMTs/NSUN1/2-targeting system for rapid prediction and in-depth analysis of drug resistance in MDS/AML. A large sized clinical study is needed to further validate its usefulness in clinical settings.