Elevated m 6 A RNA Modifications Associate with Immune Dysregulation and Cancer in People with HIV-1

(277 words) Background: N 6-methyladenosine (m 6 A) modifications of human immunodeficiency virus type 1 (HIV-1) and cellular RNA contribute to viral immune evasion and regulation of host and viral gene expression. We reported elevated RNA m 6 A levels in peripheral blood mononuclear cells (PBMCs) from HIV-1 viremic individuals compared to those on antiretroviral therapy (ART). RNA m 6 A dysregulation has been implicated in many types of cancer. However, the role of m 6 A modifications in HIV-1-associated cancers remains to be investigated. In this study, we aim to address this important question using clinical samples. Methods: We quantified RNA m 6 A levels in PBMCs from 43 de-identified people living with HIV-1 (PLWH), comparing those with cancer (n=15) to those without cancer (n=28). We used enzyme-linked immunosorbent assay (ELISA) to measure RNA m 6 A levels in PBMCs. Using an array of reverse transcription quantitative polymerase chain reaction (RT-qPCR), we performed quantitative transcriptomic analysis of 84 IFN-I-responsive genes in PBMCs. Furthermore, we performed linear regression analyses of cellular RNA m 6 A levels with HIV-1 RNA copies and CD4 + T cell counts in peripheral blood. Results: We found that m 6 A levels of PBMCs were 2.8-fold higher in the cancer group and correlated with expression of m 6 A regulatory genes. Higher m 6 A levels were also associated with increased HIV-1 RNA copies and reduced CD4 + T cell counts. HIV-1 viral load in the cancer group was higher than the non-cancer group. Transcriptomic analysis of 84 IFN-I-responsive genes revealed upregulation of many pro-inflammatory and interferon-stimulated genes in PLWH with cancer. Conclusions: Our findings suggest that HIV-1 infection and cancer microenvironment-mediated m 6 A reprogramming may contribute to chronic immune activation and malignancy in PLWH. Our results also highlight a post-transcriptional mechanism linking HIV-1 persistence to cancer risk.