Abstract 2729: Investigating the interplay between RNA methyltransferase PCIF1 and DNA hypomethylating drug decitabine in <i>TP53</i>-mutated acute myeloid leukemia

Abstract PCIF1 is an RNA methyltransferase that interacts with RNA polymerase during transcription and catalyzes N6,2'-O-dimethyladenosine (m6Am) modification on nascent mRNA, Decitabine (DAC), a DNA hypomethylating drug treating acute myeloid leukemia (AML), would activate hypermethylated genes in a way of non-specific action, which might inadvertently promote the expression of anti-apoptosis gene, leading to drug resistance. In this study, we unveil the role of the RNA modification m6Am and its writer protein PCIF1 in modulating AML's sensitivity to DAC. Utilizing KG-1a, a TP53-mutated AML cell line, we confirmed that the overexpression of catalytically active PCIF1 sensitizes leukemic cells to DAC treatment. This is evidenced by a higher apoptosis rate, increased S phase arrest observed through propidium iodide (PI) staining, and significant DNA demethylation on given genes detected via methylated DNA immunoprecipitation (MeDIP). Additionally, in terms of the second role of PCIF1 in this study, we noted elevated mRNA and protein levels of p21 in PCIF1 overexpressing cells, with p21 expression becoming even more pronounced following DAC treatment. RNA immunoprecipitation (RIP) confirmed increased direct binding of overexpressed PCIF1 to p21 mRNA. Correspondingly, there was no significant upregulation of well-established upstream regulators of p21, such as the p-Akt signaling pathway, DNA damage response cascade, or Sp1 protein. To elucidate the role of elevated p21 levels in modulating DAC's cytotoxic effects, we used Annexin-V-conjugated magnetic beads to separate dead and alive cells post-DAC treatment and found a skewed distribution of p21 in live cells. Moreover, treating PCIF1 overexpressing cells with a combination of DAC and UC2288, a p21 transcription inhibitor, proved more effective at eliminating leukemic cells than DAC alone. In short, we conclude that the second role of PCIF1 is promoting p21 expression through its catalytic function and further neutralizing the cytotoxicity effect of DAC. Nevertheless, the DAC-sensitizing pathway mediated by PCIF1 ultimately prevails, resulting in increased apoptosis in the PCIF1 overexpression model. Comparing the two axes, from a prognostic perspective for AML, high PCIF1 and low p21 expression levels appear to be more favorable. Clinical data from the TCGA LAML dataset supports this, showing significantly better survival rates in patients with high PCIF1 and low CDKN1A (the gene name of p21 protein) expression compared to those with low PCIF1 and high CDKN1A expression. These findings pave the way for clinical research to explore whether PCIF1 and p21 protein levels influence the response rate or prognosis of AML patients receiving DAC treatment. Citation Format: Li-Yang Shiau, Po-Shen Ko, Kao-Jung Chang, Shih-Hwa Chiou. Investigating the interplay between RNA methyltransferase PCIF1 and DNA hypomethylating drug decitabine in TP53-mutated acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2729.