The mechanisms governing disease-associated chromatin organization and drug selectivity are poorly understood. Our epigenetic drug screening experiments identify two distinct lineage-specific drug-responsive patterns in myeloid leukemia (MDS/AML) cells. We have established multiple MDS/AML cell lines resistant to nucleic acid analogues with different chemical backbones and have demonstrated a clear relationship between drug-resistance and sugar-phosphate backbone. Based on these results, we used various experimental approaches to elucidate leukemia-associated, drug-responsive chromatin structural changes and their underlying mechanisms. Our data demonstrate that hnRNPK, a conserved factor in heterogeneous nuclear RNA-binding protein (hnRNP) complexes, directly interacts with RNA m5C methyltransferases NSUNs/DMNT2 and RNA-polymerase II (pol-II)/CTD9 as well as erythroid vs myeloid lineage-determining transcription factors (TFs) GATA1 and SPI1/PU.1 to form distinct drug-responsive chromatin structures in MDS/AML cells. hnRNP preferentially binds to methylated polypyrimidine RNA sequences to facilitate transcription elongation. Compared to azacitidine-sensitive MDS/AML cells, there is a marked increase in azacitidine-resistant MDS/AML cells in RNA m5C and NUSNs/ DNMT2 associated with a marked increase in the interaction between hnRNPK and active pol-II/CDK9. By employing a newly-developed nascent RNA capturing technology coupled with super-resolution stimulated-emission depletion confocal microscopy, we demonstrate co-localization of hnRNPK with the TFs and active pol-II at nascent RNA sites in MDS/AML cells, further supporting the existence of hnRNPK-mediated drug-responsive transcription and lineage-specific chromatin structures in MDS/AML cells. Our experiments using clinical specimens demonstrate a positive correlation between MDS/AML progression and increase in expression of hnRNPK and RNA m5C methyltransferases, supporting their importance and clinical relevance. Furthermore, knockdown of hnRNPK and RNA 5mC methyltransferases effectively inhibited the growth of MDS/AML cells. In conclusion, our data demonstrate distinct RNA m5C methyltransferases/hnRNPK-mediated chromatin structures that control the growth and drug sensitivity of MDS/AML cells. Based on our data, we propose a novel working model of RNA m5C methyltransferase/hnRNPK-mediated drug-responsive chromatin structure in MDS/AML cells, in which RNA m5C methyltransferases function as the writers of m5C on nascent RNA, and hnRNPK functions as a reader of the RNA m5C and an operator to regulate transcription elongation and gene activation. Such novel chromatin structure-based drug action models and mechanisms may identify new diagnostic and prognostic biomarkers and therapeutic approaches.

Note: This abstract was not presented at the meeting.

Citation Format: Jason X. Cheng, Li Cheng, Adam Cloe, Yuan Li, Ming Yue, Michelle M. Le Beau, Richard A. Larson, James W. Vardiman. RNA m5C methyltransferases and hnRNPK mediate disease-associated chromatin structure and drug resistance in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4470. doi:10.1158/1538-7445.AM2017-4470