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We are analyzing https://www.biorxiv.org/content/10.1101/2025.06.24.661348v1.

Title:
5-azacytosine induces cytotoxicity via 5-methylcytosine depletion on chromatin-associated RNA in leukemia | bioRxiv
Description:
5-azacytidine (5-azaC) is a DNA hypomethylating agent clinically used to improve outcomes in myeloid malignancies. However, 5-azaC treatment causes gene dysregulation inconsistent with DNA hypomethylation changes, suggesting alternative mechanisms of action by 5-azaC. As a ribonucleoside analogue, 5-azaC is more readily incorporated into nascent RNA. Here, we demonstrate that RNA 5-methylcytosine (m5C) depletion by 5-azaC treatment, particularly at early time points, is sufficient to induce leukemia cell death. In contrast to its DNA demethylation function, the RNA-dependent effect of 5-azaC causes transcriptional repression, disrupting genes involved in cell cycle regulation and DNA repair. Mechanistically, 5-azaC impairs two specific m5C-mediated transcriptional regulatory pathways. First, depletion of m5C in chromatin-associated RNA (caRNA) disrupts the MBD6-mediated H2AK119ub deubiquitination. In parallel, this also impairs SRSF2 recruitment and the downstream H3K27ac deposition by p300. Indeed, loss of the caRNA methyltransferase NSUN2 caused prolonged cell cycle, defective DNA repair, and shifted hematopoietic lineage commitment toward erythropoiesis, mirroring the effects of 5-azaC treatment. Furthermore, we performed a leukemia cell line screen and identified that TET2 and IKZF1 depletion can sensitize 5-azaC treatment, consistent with the observed RNA-dependent cytotoxicity of 5-azaC in leukemic cells. In summary, our findings highlight the transcription repression by 5-azaC through depleting caRNA m5C, providing additional insights into the mechanism of action for 5-azaC, the prediction of its efficacy, and future directions for therapy developments based on 5-azaC. HIGHLIGHT ### Competing Interest Statement C.H. is a scientific founder, a member of the scientific advisory board and equity holder of Aferna Bio, AllyRNA, and Ellis Bio, a scientific cofounder and equity holder of Accent Therapeutics, and a member of the scientific advisory board of Rona Therapeutics and Element Biosciences. The other authors declare no competing interests. C.H. and B.G. are on a patent filed regarding the transcriptional repression caused by RNA m5C depletion of 5-azaC. Foundation for the National Institutes of Health, R01 HL174477 Cancer Prevention and Research Institute of Texas, CPRIT RP250055 Howard Hughes Medical Institute, https://ror.org/006w34k90
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12 years and 2 months (reg. 2013-04-08).

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💥 Very Strong Traffic: 200k - 500k visitors per month


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Keywords {🔍}

azac, biology, depletion, cell, rna, leukemia, dna, carna, scientific, treatment, transcriptional, repression, institute, medrxiv, subject, reviews, clinical, preprint, cytotoxicity, rnadependent, impairs, holder, cancer, biorxiv, university, trials, epidemiology, search, results, methylcytosine, chromatinassociated, pdf, summary, action, sufficient, cycle, repair, disrupts, hakub, deubiquitination, srsf, recruitment, hkac, deposition, caused, effects, tet, ikzf, highlight, azacinduced,

Topics {✒️}

epidemiology subject categories clinical trials mbd6-mediated h2ak119ub deubiquitination cell cycle regulation observed rna-dependent cytotoxicity genetics microbiology biorxiv subject category impairs srsf2 recruitment depleting carna m5c epidemiology papers author/funder reviews rna m5c depletion transcriptional repression caused keyword advanced search health-related information scientific advisory board rna-dependent effect feng-chun yang gene dysregulation inconsistent suggesting alternative mechanisms early time points disrupting genes involved providing additional insights therapy developments based generous financial support chan zuckerberg initiative imperial college london vrije universiteit amsterdam articles animal behavior dna demethylation function defective dna repair downstream h3k27ac deposition impeding p300 recruitment context sensitize 5-azac treatment ikzf1 depletion org/006w34k90 copyright 5-azac impairs h2ak119ub deubiquitination national institutes research institute 5-methylcytosine depletion carna transcriptional repression dna repair scientific founder scientific cofounder disrupts

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