Elucidating the role of retrotransposon-mediated viral mimicry in leukemia therapy

Abstract Hypomethylation agents (HMA), including 5-azacytidine (AZA) and decitabine (DAC), are frontline therapies for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). HMA-induced DNA hypomethylation can lead to the reactivation of tumor suppressor genes, thereby impairing leukemia cell proliferation and survival. However, the broader transcriptional impact of HMA treatment, particularly on epigenetically silenced genomic repetitive elements, and their contribution to therapeutic efficacy remain largely unexplored. Retrotransposable elements (RTEs), also known as retroelements or retrotransposons, are repetitive genomic parasites that play pivotal roles in genome regulation and immune modulation. We hypothesize that HMA-mediated reactivation of RTEs triggers an antiviral-like immune response, or ‘viral mimicry’, in leukemia cells, contributing to the therapeutic efficacy of hypomethylating agents. To test this, we performed transcriptomic and epigenomic profiling of four representative pediatric myeloid leukemia cell lines at multiple time points (day 1, 4, and 7) following 500 nM of AZA or DAC treatment. We developed computational pipelines to annotate and quantify RTE expression and identified a subset of RTEs, including endogenous retroviruses (ERVs), LINE-1, and SINE, showing cell type-, oncogenotype-, and treatment-specific reactivation. RTE reactivation was linked to dysregulation of immune gene expression, cellular stress, and apoptosis. Notably, innate immune response genes, including interferon-stimulated genes (e.g., IFNB1 and ISG15), cytosolic double-stranded RNA sensors (e.g., IFIH1/MDA5 and RIG-I/DDX58), and proinflammatory cytokines (e.g., IL1B andIL18), were significantly upregulated. Furthermore, by analyzing genomic locus-specific RTE expression, we uncovered distinct molecular features, such as epigenetic state, transcription factor occupancy, and differentiation status, that can explain HMA-mediated effects on RTE reactivation. To evaluate the role of RTE-mediated viral mimicry response in HMA therapy for AML, we analyzed RNA-seq data from the AML16 clinical cohort, comprising 187 pediatric AML patients treated with AZA or DAC at St. Jude Children's Research Hospital. We identified RTE reactivation and immune gene dysregulation following HMA treatment, with patterns correlating to specific molecular subtypes. To quantify this response, we developed a ‘viral mimicry score’ (VMS), capturing RTE-mediated antiviral activation in individual patients. VMS analysis revealed marked inter-patient variability in RTE reactivation and immune pathway induction, highlighting the heterogeneous immune-epigenetic landscape of myeloid leukemia and the potential of VMS to stratify patients by sensitivity to epigenetic therapy. Applying the VMS to independent, published cohorts of HMA-treated chronic myelomonocytic leukemia (CMML), MDS and AML patients revealed that higher scores were associated with better treatment response, highlighting the potential role of viral mimicry in mediating therapeutic efficacy of epigenetic therapies in myeloid malignancies.Together, these findings not only uncover the molecular basis of HMA-induced transcriptional response in leukemia but also establish the RTE-driven viral mimicry as a potential biomarker for hypomethylation therapy. Further investigation may enable its application in patient stratification, treatment monitoring, and prognostic assessment in hematologic malignancies.