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

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Stapleton, Li Wu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8070380/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Mar, 2026 Read the published version in BMC Cancer → Version 1 posted 11 You are reading this latest preprint version Abstract (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. HIV-1 infection cancer people living with HIV-1 (PLWH) N6-methyladenosine (m6A) peripheral blood mononuclear cells type-I interferon (IFN-I) IFN-responsive genes antiretroviral therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background The eukaryotic RNAs have more than 170 types of modifications, among which N 6 -methyladenosine (m 6 A) is the most abundant internal modification in mRNA and various non-coding RNAs [ 1 , 2 ]. m 6 A modification influence RNA metabolism by regulating splicing, nuclear export, translation efficiency and transcript stability [ 2 , 3 ]. The dynamic m 6 A modifications are installed by writer enzymes [methyltransferase like 3/14 (METTL3/14) and Wilms tumor 1-associated protein (WTAP)], removed by erasers [AlkB homolog 5 (ALKBH5) and fat mass and obesity-associated protein (FTO)], and interpreted by reader proteins [YTH domain-containing family 1–3 (YTHDF1-3), collectively called as m 6 A regulatory machinery [ 4 ]. The dysregulation of RNA m 6 A modification and its regulatory machinery, including writers, erasers and readers are frequently observed in pathogenesis of various diseases including viral infections and cancers [ 5 – 7 ]. m 6 A modifications have been found on both HIV-1and host transcripts, depicting an intricate role for epitranscriptomic regulation in viral replication, immune evasion, and latency reactivation [ 8 – 12 ]. HIV-1 not only exploits the host m 6 A methylation machinery to enhance its gene expression and replication, but also modulates the expression of m 6 A regulators, leading to broader effects on host immune pathways and gene expression [ 7 ]. Our previous study showed that combined antiretroviral therapy (ART) significantly suppresses RNA m 6 A levels in peripheral blood mononuclear cells (PBMCs) from people living with HIV-1 (PLWH) [ 13 ]. Despite the success of ART, PLWH exhibit a significantly increased risk of developing certain cancers, including Kaposi’s sarcoma, non-Hodgkin lymphoma, and cervical cancers [ 14 , 15 ]. This increased cancer risk is partially attributed to chronic immune activation, persistent inflammation, co-infections with oncogenic viruses, and impaired immune surveillance [ 16 ]. However, the contribution of RNA modifications, especially m 6 A methylation to oncogenesis in PLWH remains underexplored. Previous studies have shown the oncogenic roles of several m 6 A regulatory proteins, such as METTL3, METTL14, FTO and YTHDF2 proteins, in promoting proliferation, metastasis and resistance to apoptosis across various cancer types [ 5 , 17 – 19 ]. Given the dual role of m 6 A in modulating antiviral immunity and cancer-associated gene expression, it is plausible that aberrant m 6 A signaling may serve as a mechanistic bridge between chronic HIV-1 infection and cancer development. Notably, m 6 A also regulates the expression and function of interferon-stimulated genes (ISGs), which are central to antiviral defense but can paradoxically promote tumorigenesis under chronic activation [ 20 ]. This intersection raises the possibility that chronic HIV-1-associated immune activation, combined with epitranscriptomic dysregulation, may influence cancer susceptibility through sustained interferon (IFN) signaling and ISG modulation. In this study, we investigated the m 6 A RNA methylations in PBMCs from 43 de-identified aging PLWH with or without cancer at average age of 56 and 60, respectively. We found significantly elevated RNA m 6 A levels in PLWH with cancer, which were accompanied by upregulated expression of mRNA encoding the m 6 A writer complex and reader YTHDF1-3 proteins. Furthermore, we observed that the cellular RNA m 6 A levels correlated with higher HIV-1 RNA copy numbers and lower CD4 + T cell counts. Importantly, we demonstrated that cancer development in PLWH was associated with aberrant expression of IFN-I-responsive genes, suggesting a link between m 6 A-modulated immune responses and oncogenesis. These findings provide new insights into how m 6 A RNA modifications may contribute to the dysregulation of innate immunity and the development of HIV-1-associated cancers. Methods Study participants and de-identified PBMCs samples PBMCs were obtained from a total of 43 PLWH with cancer (n = 15) or without cancer (n = 28) and de-identified sample details were described in Tables 1 and 2 , respectively. Thirteen of the de-identified samples (n = 13) were provided by the AIDS and Cancer Specimen Resource funded by the National Cancer Institute, NIH. Individuals (n = 30) attending the University of Iowa Virology Clinic were invited to participate in this study and, following written informed consent, provided blood samples. The informed consent to participate was obtained from all of the participants in the study. PMBCs were collected from PLWH with a documented history of cancer, or from those without cancer. Blood samples were obtained for the preparation of PBMCs as described [ 21 ]. Medical records of those with HIV-1 were reviewed. CD4 + T cells count was either obtained at the same visit, or the most recent (within 2 years) in those with over 2 years viral load suppression, and HIV-1 viral load were documented. Plasma HIV-1 viral load was conducted using the COBAS® AmpliPrep/COBAS® TaqMan HIV-1 test (Roche). PBMCs were preserved in liquid nitrogen until use. Statistical analyses of the age, HIV-1 viral load, and CD4 + T cells counts between the cancer and non-cancer groups are shown in Fig. 1 . Table 1 Details of PBMC samples from de-identified PLWH with cancer Participant Age Gender HIV-1 viral load (RNA copies/mL) CD4 + T cell count/mm 3 blood Cancer type a Cancer stage Treatment stage and type ART regimen Drugs of abuse Marijuana use 1 b 40 M c 115,037 24 KS d High Risk None None Unknown Unknown 2 45 M 67,633 236 KS Low Risk None None Unknown Unknown 3 46 M 99,139 195 KS Low Risk None None Unknown Unknown 4 45 M 54,766 31 KS High Risk None None Unknown Unknown 5 55 M 17,078 14 KS High Risk None None Unknown Unknown 6 40 M 248,068 64 KS High Risk None None Unknown Unknown 7 48 M 400 223 KS Low Risk ART ~ 2.5 mos prior to sample collection lopinavir/ritonavir and emtricitabine/ tenofovir Unknown Unknown 8 e 82 M 148 606 Prostate cancer Remission Prostatectomy 2006 Biktarvy No No 9 54 F f S g 1,497 Hodgkin’s disease Remission Splenectomy, 1980s Biktarvy Yes, many Yes 10 68 M S 885 Non-melanocytic skin cancer Cured Surgical removal Biktarvy No Yes 11 59 M 30 262 KS Remission Diagnosed 2012- responded to ART Biktarvy Yes No 12 69 M 139 506 Large B cell lymphoma Remission Treated with RCHOP 2023 Biktarvy No Yes 13 60 F S 941 Vulvar cancer, Anal squamous cell x 3 remission, active Resected 2022, lymph node resection, XRT, Resected 2024–2025 Juluca No No 14 58 M S 703 Anal dysplasia Removed Resected 2016 Biktarvy No No 15 68 F 11,500 718 Lung cancer Resected Adenocarcinoma, resected and XRT 2022–2023 Biktarvy Yes, remote No Mean ± SD 56 ± 12 55,813 ± 72,821 460 ± 414 Only de-identified information is shown in the table. The informed consent to participate was obtained from all of the participants. a Cancer present or being treated at time of blood acquisition. b Samples from participants 1–7 were provided by the AIDS and Cancer Specimen Resource funded by the National Cancer Institute, NIH; Cancer stage is based on ACTG Staging for KS using tumor extent, immune status, and systemic illness factors. c M: male. d KS: Kaposi sarcoma. e Samples from participants 8–15 were provided by the University of Iowa HIV Clinic. f F: female. g S: HIV-1 RNA suppressed (viral load ≤ 20 copies/mL). Table 2 Details of PBMC samples from de-identified PLWH without cancer Participant ID Age Gender HIV-1 viral load (RNA copies/mL) CD4 + T cell count/mm 3 blood ART regimen Drugs of abuse Marijuana use 1 a 54 M b 40 511 Lamivudine/Zidovudine/Nevirapine No No 2 52 M 60,785 154 None No No 3 61 F c 100 428 Lamivudine/Zidovudine/Nevirapine No No 4 55 M 100 485 Lamivudine/Zidovudine/Efavirenz No No 5 61 F 66,714 337 None No No 6 62 M 112,030 211 None No No 7 d 60 M S e 661 Biktarvy Yes - many Yes 8 53 M 35 338 Genvoya No Yes 9 68 M S 1,400 Dovato No Yes 10 66 M S 267 Juluca No Yes 11 65 M S 749 Triumeq No No 12 61 M S 790 Descovy + Tivicay No No 13 60 M 39 130 Biktarvy Yes Yes 14 58 F S 1,667 Cabenuva No No 15 57 M S 835 Dovato No No 16 55 M 133 1,059 Dovato Yes Yes 17 71 F S 1,078 Biktarvy Yes Yes 18 53 M S 338 Genvoya No Yes 19 67 M 65 604 Biktarvy No No 20 50 M 64 846 Biktarvy No No 21 61 M 31,500 147 Biktarvy Yes Yes 22 50 M 105 235 Biktarvy No No 23 60 F 28 634 Descovy + Tivicay No No 24 60 M S 832 Biktarvy No No 25 64 M S 620 Biktarvy No Yes 26 61 M 23 460 Odefsey No Yes 27 60 M S 1,343 Biktarvy Yes No 28 60 M 296 182 Biktarvy No Yes Mean ± SD 60 ± 5 17,004 ± 32,656 619 ± 399 Only de-identified information is shown in the table. The informed consent to participate was obtained from all of the participants. a Samples from participants 1–6 were provided by the AIDS and Cancer Specimen Resource funded by the National Cancer Institute, NIH. b M: male. c F: female. d Samples from participants 7–28 were provided by the University of Iowa HIV Clinic. e S: HIV-1 RNA suppressed (viral load ≤ 20 copies/mL). Cellular RNA isolation and RT-qPCR Total RNA isolation was performed using TRIzolT M reagent (ThermoFisher Scientific, 15596018) following manufacturers guidelines. The isolated RNA was quantified using a NanoDrop OneC spectrophotometer (ThermoFisher Scientific). DNase-treated RNA (500 ng) served as a template for cDNA synthesis, using the iScript cDNA Synthesis Kit following the manufacturer’s protocol (Bio-Rad, 1708890). qRT-PCR was performed with the iTaqSYBR Green PCR Kit (#1725124, Biorad) in the BioRad CFX96 Real-Time PCR system to evaluate the relative mRNA expression of the m 6 A writer complex genes ( METTL3 , METTL14 , WTAP , RBM15 , and VIRMA ), erasers ( ALKBH5 and FTO ), and readers ( YTHDF1-3 ) in PBMCs obtained from PLWH with cancer and without cancer. All reactions were performed in triplicate and normalized with GAPDH as a housekeeping gene. The relative gene expression of each sample was calculated using the 2 −ΔΔ C t formula. The primers for qRT-PCR were described in our previous study [ 13 ]. RNA mA quantification by ELISA RNA m 6 A ELISA was performed according to the published protocol [ 12 , 13 ]. Briefly, 200 ng of total RNA per sample was quantified in triplicate. The primary m 6 A antibody solution (ABclonal, A19841) was prepared by diluting 1:10,000 in PBST (PBS with 0.1% Tween 20), and the secondary anti-rabbit IgG solution (Promega, W4011) was prepared by diluting 1:5000 in PBST. Each well was sequentially incubated with 100 µL of primary antibody solution, followed by 100 µL of secondary antibody solution. The signal was developed using 100 µL of 3,3′,5,5′-tetramethylbenzidine substrate (ThermoFisher Scientific, 34021) and quenched with 100 µL of stop solution (2% H 2 SO 4 ). Absorbance was measured at 450 nm, and m 6 A levels were quantified relative to a standard curve generated using defined concentrations of m 6 A-modified RNA (EpiGentek, P-9005-96-PC). Results Elevated RNA m 6 A levels in PBMCs from de-identified PLWH with cancer To examine the potential effects of HIV-1 infection and cancer status on cellular RNA m 6 A levels ex vivo , we obtained PBMCs from 43 de-identified PLWH, including those with various types of cancer (n = 15) and without cancer (n = 28). The informed consent to participate was obtained from all of the participants in the study. The age, gender, HIV-1 plasma viral load, and CD4 + T cell counts, cancer type, treatment stage and type, ART regimen, drugs of abuse, use of marijuana of de-identified PLWH with cancer are summarized in Table 1 . The age, gender, HIV-1 plasma viral load, CD4 + T cell counts, ART regimen, drug of abuse, and use of marijuana of de-identified PLWH without cancer are summarized in Table 2 . Only de-identified information is provided in Tables 1 – 2 . Statistical analyses indicated that HIV-1 viral load was higher in participants in the cancer group compared to those in the non-cancer groups, while the age and CD4 + T cell counts did not show statistical differences between the two groups (Fig. 1 ). Relative m 6 A levels in RNA from PBMCs of PLWH were quantified using m 6 A ELISA [ 13 ]. PLWH with cancer exhibited approximately 2.8-fold higher m 6 A levels than those without cancer (p ≤ 0.01) (Fig. 2 ). The increased m 6 A levels are also correlated with higher viral load in the cancer group compared to non-cancer group (Fig. 1 ). This observation extends our previous findings that viremic individuals show elevated m 6 A levels compared to PLWH on ART [ 13 ]. These findings suggest a potential link between HIV-1 infection and elevated m 6 A levels, which may contribute to dysregulation of host gene expression and possibly increase the risk of cancer development in PLWH. Correlation of cellular RNA mA levels with HIV-1 viral load and CD4 T cell counts To determine whether RNA m 6 A levels are associated with HIV-1 replication, we analyzed the correlation between m 6 A levels in PBMCs and plasma HIV-1 RNA copies of PLWH. In the combined dataset (n = 43), cellular m 6 A RNA levels showed a significant positive correlation with HIV-1 RNA levels (p = 0.0005; Fig. 3 A). When analyzed separately, a significant negative correlation was observed between m 6 A levels and HIV-1 RNA copies in PLWH without cancer (n = 28, p = 0.0082; Fig. 3 B), whereas a strong positive correlation was found in PLWH with cancer (n = 15, p = 0.0059; Fig. 3 C). These data demonstrate that elevated HIV-1 viral load and cancer tumor microenvironment is associated with increased RNA m 6 A levels in PBMCs, suggesting that HIV-1 viral load and tumor microenvironment in PLWH with cancer enhance cellular RNA m 6 A modifications. We also assessed the association between cellular m 6 A RNA levels and CD4 + T cell counts of PLWH. In the overall cohort, a significant negative correlation was observed (n = 43, p = 0.001; Fig. 3 D). This trend remained significant among PLWH with cancer (n = 15, p = 0.02; Fig. 3 F), but not in PLWH without cancer (n = 28, p = 0.2725; Fig. 3 F). The increased m 6 A levels and the association with CD4 + T cell decline in PLWH with cancer may also contribute to heightened cancer risk in PLWH [ 22 , 23 ]. Expression of m 6 A regulators genes in PBMCs from PLWH with or without cancer To determine whether elevated m 6 A RNA levels in PLWH with cancer are driven by altered expression of m 6 A regulatory genes, we measured the mRNA levels of major genes encoding m 6 A writers, erasers, and readers (Fig. 4 ). We observed a significant increase in the expression of the core methyltransferase components METTL3, METTL14, as well as the accessory proteins RBM15 and VIRMA, in PBMCs from PLWH with cancer compared to those without cancer (Fig. 4 A). In contrast, WTAP, an essential scaffolding subunit of the methyltransferase complex, showed no change in expression, suggesting that its regulatory function may be modulated post-transcriptionally or through other mechanisms [ 24 ]. In the m 6 A erasers, we detected a modest increase in ALKBH5 mRNA expression, while FTO mRNA expression remained unchanged according to statistical analysis (Fig. 4 B). The mRNA levels of the m 6 A reader proteins YTHDF1-3 were significantly elevated in PLWH with cancer compared to those without cancer (Fig. 4 C). These findings suggest that the increased m 6 A RNA methylation levels may be driven by transcriptional upregulation of m 6 A regulatory genes. However, it is important to note that mRNA expression levels do not always correspond to protein abundance or enzymatic activity, and further studies are needed to assess protein-level changes and post-translational regulation. Dysregulated expression of IFN-I-responsive genes in PLWH with cancer Given the functional significance of m 6 A modification and importance of IFN-I-associated genes in regulating both HIV-1 infection and cancer [ 7 , 11 , 25 ], we explored the expression of IFN-I-responsive genes in PLWH with cancer compared to individuals without cancer. Using an RT-qPCR array we analyzed the mRNA levels of 84 IFN-I-responsive genes, including interferons, signaling molecules, receptors, ISGs, and IFN-resistance markers. The assay allowed us to estimate the RNA levels of 84 IFN-I associated genes in one plate with respect to five housekeeping genes (Fig. 5 A and 5 B). Heat maps were generated to depict the average change in gene expression in PLWH with cancer (n = 15) or without cancer (n = 28). Overall, many IFN-I-responsive genes expression levels were upregulated in caner-positive with respect to cancer-negative PLWH (Fig. 5 B). Significantly regulated IFN-I-responsive gene expression in PLWH with cancer Using the IFN-I-responsive gene expression array, we identified a panel of genes significantly upregulated in PLWH with cancer (Fig. 6 A). A comparative analysis of the top ten upregulated genes (based on fold change) between cancer-positive and cancer-negative PLWH revealed a distinct transcriptional signature (Fig. 6 A). Among these, MET proto-oncogene, receptor tyrosine kinase ( MET ) and IFN-alpha-inducible protein 27 ( IFI27 ) mRNA levels were the most significantly elevated in cancer-positive PLWH, exhibiting ~ 32-fold (p = 0.0001) and ~ 14-fold (p = 0.0001) increases, respectively. This observation is consistent with our prior findings that HIV-1 infection induces MET and IFI27 expression in viremic PLWH compared to HIV-1-suppressed individuals on ART [ 13 ]. Although the role of IFI27 in HIV-1 infection and progression have been reported [ 13 ], its function in context of oncogenesis in PLWH remains to be explored. MET is involved in cell proliferation, survival, and metastasis [ 26 ], and its overexpression has been implicated in various cancers [ 27 ], suggesting that its upregulation in the HIV-1 and cancer context may contribute to tumorigenesis. IFI27, a well-known ISG, has been implicated in both viral infection responses [ 28 ] and cancer progression through its regulation of apoptosis and immune evasion [ 29 , 30 ]. Several other IFN-I-responsive genes were also significantly upregulated in cancer-positive PLWH, including ISG15 , C-X-C motif chemokine ligand 10 ( CXCL10 ), interferon-induced protein with tetratricopeptide repeats 3 ( IFIT3 ), interleukin 6 ( IL6 ), interferon regulatory factor 7 ( IRF7 ), interferon-induced transmembrane protein 3 ( IFITM3 ), interferon-gamma inducible protein 16 ( IFI16 ), and signal transducer and activator of transcription 1 ( STAT1 ). Furthermore, three innate immunity regulatory genes were significantly downregulated (p < 0.05) in cancer-positive PLWH compared to cancer-negative PLWH, including genes encoding interferon regulatory factor 5 ( IRF5 ), Toll-like receptor 7 ( TLR7 ), and TLR8 (Fig. 6 B). These genes are known to contribute to chronic immune activation, inflammation, and tumor-promoting immune landscapes [ 31 ]. Together, these data suggest a dynamic reprogramming of IFN-I signaling in PLWH with cancer, potentially driven by chronic infection and altered m 6 A-mediated gene regulation. Discussion The epitranscriptomic m 6 A RNA modification is the most prevalent internal modification on eukaryotic mRNA and is dynamically regulated by a set of proteins known as m 6 A regulators controlling the fate of RNA. We found that RNA m 6 A levels of PBMCs from PLWH with cancer were higher compared to PBMCs from PLWH without cancer. This suggest that cancers further enhance m 6 A RNA modification in PLWH. These observations align with previous reports [ 32 ] that enhanced m 6 A modification levels can serve as a biomarker for both HIV-1 infection and cancer progression. To explore the connection between m 6 A RNA modification, HIV-1 replication, and cancer status, we performed correlation analysis. We observed a positive correlation between m 6 A levels and HIV-1 RNA copies when data from all PLWH were analyzed collectively (Fig. 3 A), supporting previous findings that HIV-1 infection and treatment with recombinant HIV-1 gp120 enhances cellular RNA m 6 A levels [ 33 ]. Interestingly, when stratified by cancer status, a positive correlation between m 6 A and viral load was observed in PLWH with cancer, whereas a negative correlation was found in PLWH without cancer. The negative correlation may be influenced by lower average plasma HIV-1 RNA copies in the non-cancer group (mean = 55,813 copies/mL) compared to cancer group (mean = 17,004 copies/mL). The smaller sample size may skew the analysis outcome. Performing the correlation analysis on a large cohort can further support the findings. We observe a significant negative correlation in CD4 + T cell counts and m 6 A RNA levels in combined dataset (n = 43) and in cancer subgroup, while a non-significant trend was observed in non-cancer subgroup. Elevated m 6 A levels might be associated with reduced CD4⁺ T cell recovery in PLWH, particularly in those with cancer. Indeed, individuals with cancer had markedly lower average CD4⁺ counts (460 ± 414) compared to those without cancer (619 ± 399). These findings emphasize the importance of early HIV-1 diagnosis and regular monitoring of both CD4⁺ T cell counts and m 6 A RNA levels to assess cancer risk in PLWH [ 16 , 22 , 34 , 35 ]. One limitation in our study is potential confounding variables in the cohort, such as HIV-1 viral load, CD4 + T cell counts, ART adherence, and drug use. Our statistical analyses indicate that the difference in the age and CD4 + T cell counts is not significant between the cancer and non-cancer groups, while the viral load is significantly higher in the cancer group compared to the non-cancer group ( p < 0.05). Our previous study showed significant higher RNA m 6 A levels in PBMCs from HIV-1 viremic individuals compared to those on ART [ 13 ]. Thus, the higher viral load in the cancer group might contribute to the increased m 6 A RNA levels compared to the non-cancer group. Notably, in the cancer cohort, 6 out of 15 PLWH had not received ART, while in the non-cancer cohort, only 3 out of 28 PLWH had not undergone ART. Therefore, the higher viral load observed in the cancer group may be partly due to the larger proportion of individuals not receiving ART. However, we believe that our results are not solely explained by HIV-1 infection and ART status but are also influenced by the cancer status of the individuals. Because we observed elevated levels of m 6 A RNA modification in PLWH with cancer compared to PLWH without cancer (Fig. 2 ), we hypothesized that this increase might be due to altered expression of m 6 A regulatory machinery, including writers, erasers, and readers. We thus analyzed the expression of key m 6 A regulatory genes. We observed a significant upregulation of METTL3 and METTL14 transcripts, the two catalytic components of the m 6 A methyltransferase complex, in cancer samples. Interestingly, mRNA expression of WTAP, a key regulatory subunit that stabilizes and scaffolds the methyltransferase complex [ 36 ], remained unchanged (Fig. 4 A). This raises the possibility that post-transcriptional or post-translational modifications (PTMs) may regulate WTAP function in HIV-1-associated cancer, requires further studies. We also observed increased expression of RBM15 that guide m 6 A deposition at specific transcript regions. The upregulation of ALKBH5 mRNA expression was unexpected and suggests a more complex regulatory mechanism that might involve substrate specificity, cellular localization, or PTMs. Due to the limited number of available cells, we could not assess protein expression levels in this study. However, our previous work indicated no significant differences in protein levels of these regulators between PLWH and healthy controls [ 33 ]. Thus, the observed increase in m 6 A levels in cancer samples cannot be solely attributed to RNA or protein abundance. It is possible that m 6 A regulatory enzyme activity is modulated by PTMs which are known to influence both writer and eraser functions [ 37 , 38 ]. Whether such PTM-based modulation contributes to elevated m 6 A levels in PLWH with cancer remains an open question and merits further investigation. We also found significant mRNA upregulation of YTHDF1-3 , which encode major members of the YTHDF family of m 6 A reader proteins, in samples from PLWH and cancer (Fig. 4 C). These proteins bind m 6 A-modified transcripts and regulate their translation or decay. Previous studies have linked YTHDF proteins to tumorigenesis in various cancers, including hepatocellular carcinoma, leukemia, and glioblastoma [ 39 ]. For instance, YTHDF2 promotes degradation of pro-apoptotic or differentiation-associated mRNAs, while, YTHDF1 enhances the translation of growth-promoting mRNAs and has been shown to correlate with poor prognosis in several malignancies. The elevated expression of YTHDF readers may reflect a cellular adaptation to chronic immune activation, viral persistence, or altered m 6 A dynamics induced by HIV-1 or ART. It remains to be determined whether these changes contribute functionally to tumor development or represent a compensatory mechanism in response to altered m 6 A dynamics. To further explore how m 6 A dysregulation intersects with immune signaling, we analyzed the expression profiles of 84 IFN-I-responsive genes and immune-related genes associated with HIV-1 pathogenesis and cancer progression. We found that mRNA levels of a subset of IFN-I responsive genes, including MET, IFI27, ISG15, CXCL10, IFIT3, IL6, IRF7, IFITM3, IFI16 , and STAT1 , were significantly upregulated in cancer samples from PLWH (Fig. 6 A). These genes and their encoded proteins play central roles in regulating antiviral immunity and inflammation. Of note, persistent expression of ISGs in PLWH, even under ART, suggests a state of chronic immune activation [ 40 ]. Importantly, many of these genes also have established roles in tumorigenesis. For instance, MET is a proto-oncogene implicated in tumor proliferation and metastasis [ 41 ]; IL6 is a pro-inflammatory cytokine that supports tumor growth and immune evasion [ 42 ]; and STAT1, though typically antiviral, has context-dependent functions in promoting or suppressing tumorigenesis [ 43 ]. Additionally, ISG15 and IFI27 modulate apoptosis and immune responses and are upregulated in various cancers and viral infections [ 44 , 45 ]. Notably, several of these transcripts such as STAT1, ISG15 , and CXCL10 have been reported to be regulated by m 6 A modification [ 20 , 46 , 47 ]. This suggests that the m 6 A RNA modification may further shape the chronic inflammatory state observed in these PLWH. A recent study showed that inhibition of METTL3 stimulates IFN-I response by promoting double stranded RNA formation and promoting anti-tumor immunity [ 48 ]. Conversely, we observed significant downregulation of IFN-I regulatory or pattern recognition receptor genes, such as IRF5, TLR7 and TLR8 in PLWH with cancer. These genes are vital for sensing viral RNA and activating IFN-I responses [ 49 , 50 ]. Their reduced expression may represent a mechanism of immune evasion or exhaustion in the tumor microenvironment, potentially contributing to impaired antiviral surveillance and increased cancer susceptibility in PLWH. Due to limited RNA availability from PBMCs in this study, we were unable to assess m 6 A methylation status of individual IFN-I-responsive transcripts. Collection of additional samples will be necessary to determine m 6 A levels and further elucidate its role in regulating RNA stability and translation in aging PLWH with cancer. Inflammation and immunosuppression induced by chronic virus infection and cancer can lead to a dysfunctional immune state [ 16 , 31 ]. IFN-I-responsive transcripts might play an important role and exert their opposing effects during cancer progression and HIV-1 infection. This potential dual role needs further investigation in future studies. We believe that the interplay between HIV-1 infection, cancer, and interferon responses is highly complex and likely influenced by multiple, overlapping factors. It is difficult to conclusively determine whether the elevated IFN-I responses observed in our dataset are driven primarily by the presence of cancer, HIV-1 infection, or their combined effect. Importantly, our prior work has shown that the HIV-1 envelope protein gp120 can upregulate m 6 A RNA modification in host cells independently of active viral replication [ 33 ]. Additionally, IFN-I transcripts themselves are subject to m 6 A modification, which targets them for degradation and helps fine-tune immune homeostasis [ 20 ]. Furthermore, HIV-1 intron-containing RNAs have been shown to activate innate immune sensors and stimulate IFN-I responses. These studies support the possibility that HIV-1 infection can modulate both m 6 A dynamics and IFN-I responses through multiple mechanisms, independent of or in conjunction with cancer. Further functional studies are required to understand the complex mechanism undying the connection between HIV-1 infection, m 6 A RNA modification, and caner progression. Conclusions Collectively, our findings suggest a complex immune landscape in HIV-1-associated cancers characterized by simultaneous overactivation of ISGs and suppression of innate sensing pathways. The interplay between m 6 A modifications and dysregulated interferon signaling may represent a critical axis in the development of HIV-1-associated cancer in aging populations. Abbreviations HIV-1 human immunodeficiency virus type 1 PLWH people living with HIV-1 m 6 A N 6 -methyladenosine PBMCs peripheral blood mononuclear cells ART antiretroviral therapy IFN-I type I interferon ELISA enzyme-linked immunosorbent assay RT-qPCR reverse transcription quantitative polymerase chain reaction ISGs interferon-stimulated genes METTL3/14 methyltransferase like 3/14 WTAP Wilms tumor 1-associated protein ALKBH5 AlkB homolog 5 FTO fat mass and obesity-associated protein YTHDF1-3 YTH domain-containing family 1–3 Declarations Ethics approval and consent to participate The study using human PBMCs from PLWH with and without cancer and consent to participate have been approved by the Institutional Review Board (IRB) of the University of Iowa. Individuals attending the University of Iowa Virology Clinic were invited to participate in this study and, following written informed consent, provided blood samples. The informed consent to participate was obtained from all of the participants in the study. The study was conducted according to the Declaration of Helsinki guidelines. Consent for publication Consent for publication has been obtained from the study participants through the approved IRB protocol as described above. Only de-identified information is shown in the sample summary Tables 1–2. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the U.S. National Institutes of Health (NIH) grants P30CA086862 (supplement 24S1 and 25S1) and R61/R33AI169659 to L.W, and in part by the Veterans Administration Health Service grant BX000207 to J.T.S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. Author Contribution Conceptualization: T.M., S.T., and L.W. Resources: J.T.S. and L.W. Methodology and Investigation: T.M. and S.T. Formal analysis: T.M., S.T., and L.W. Writing-Original Draft: T.M. and S.T., and L.W. Writing-Reviewing and Editing: J.S. and L.W. Visualization: T.M. and L.W. Supervision, Project administration, and Funding acquisition: L.W. All authors read and approved the final manuscript. Acknowledgement The authors thank Ms. Bethany Wilms and Mr. Ashok Chaudhary for technical assistance and the Wu lab members for helpful discussion and suggestions. Thirteen de-identified specimens in this study (detailed in Tables 1-2 with de-identified information) were provided by the AIDS and Cancer Specimen Resource, funded by the National Cancer Institute, NIH. Data Availability All data generated or analyzed during this study are included in this manuscript for publication. References Roundtree IA, Evans ME, Pan T, He C. Dynamic RNA Modifications in Gene Expression Regulation. Cell. 2017;169(7):1187–200. Zhao BS, Roundtree IA, He C. Post-transcriptional gene regulation by mRNA modifications. Nat Rev Mol Cell Biol. 2017;18(1):31–42. Meyer KD, Jaffrey SR. The dynamic epitranscriptome: N6-methyladenosine and gene expression control. Nat Rev Mol Cell Biol. 2014;15(5):313–26. He PC, He C. m(6) A RNA methylation: from mechanisms to therapeutic potential. EMBO J. 2021;40(3):e105977. Barbieri I, Kouzarides T. Role of RNA modifications in cancer. Nat Rev Cancer. 2020;20(6):303–22. Zhang Z, Liu F, Chen W, Liao Z, Zhang W, Zhang B, Liang H, Chu L, Zhang Z. The importance of N6-methyladenosine modification in tumor immunity and immunotherapy. Exp Hematol Oncol. 2022;11(1):30. Phillips S, Mishra T, Huang S, Wu L. Functional Impacts of Epitranscriptomic m(6)A Modification on HIV-1 Infection. Viruses 2024, 16(1). Tirumuru N, Zhao BS, Lu W, Lu Z, He C, Wu L. N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression. eLife 2016, 5. Lichinchi G, Gao S, Saletore Y, Gonzalez GM, Bansal V, Wang Y, Mason CE, Rana TM. Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells. Nat Microbiol. 2016;1:16011. Lu W, Tirumuru N, St Gelais C, Koneru PC, Liu C, Kvaratskhelia M, He C, Wu L. N(6)-Methyladenosine-binding proteins suppress HIV-1 infectivity and viral production. J Biol Chem. 2018;293(34):12992–3005. Chen S, Kumar S, Espada CE, Tirumuru N, Cahill MP, Hu L, He C, Wu L. N6-methyladenosine modification of HIV-1 RNA suppresses type-I interferon induction in differentiated monocytic cells and primary macrophages. PLoS Pathog. 2021;17(3):e1009421. Mishra T, Phillips S, Zhao Y, Wilms B, He C, Wu L. Epitranscriptomic m(6)A modifications during reactivation of HIV-1 latency in CD4(+) T cells. mBio. 2024;15(11):e0221424. Mishra T, Phillips S, Maldonado C, Stapleton JT, Wu L. Antiretroviral Therapy Suppresses RNA N(6)-Methyladenosine Modification in Peripheral Blood Mononuclear Cells from HIV-1-Infected Individuals. AIDS Res Hum Retroviruses. 2024;40(9):511–20. Shiels MS, Pfeiffer RM, Gail MH, Hall HI, Li J, Chaturvedi AK, Bhatia K, Uldrick TS, Yarchoan R, Goedert JJ, et al. Cancer burden in the HIV-infected population in the United States. J Natl Cancer Inst. 2011;103(9):753–62. Chao C, Leyden WA, Xu L, Horberg MA, Klein D, Towner WJ, Quesenberry CP Jr., Abrams DI, Silverberg MJ. Exposure to antiretroviral therapy and risk of cancer in HIV-infected persons. AIDS. 2012;26(17):2223–31. Hernandez-Ramirez RU, Shiels MS, Dubrow R, Engels EA. Cancer risk in HIV-infected people in the USA from 1996 to 2012: a population-based, registry-linkage study. Lancet HIV. 2017;4(11):e495–504. Lin S, Choe J, Du P, Triboulet R, Gregory RI. The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells. Mol Cell. 2016;62(3):335–45. Weng H, Huang H, Wu H, Qin X, Zhao BS, Dong L, Shi H, Skibbe J, Shen C, Hu C, et al. METTL14 Inhibits Hematopoietic Stem/Progenitor Differentiation and Promotes Leukemogenesis via mRNA m(6)A Modification. Cell Stem Cell. 2018;22(2):191–205. e199. Paris J, Morgan M, Campos J, Spencer GJ, Shmakova A, Ivanova I, Mapperley C, Lawson H, Wotherspoon DA, Sepulveda C, et al. Targeting the RNA m(6)A Reader YTHDF2 Selectively Compromises Cancer Stem Cells in Acute Myeloid Leukemia. Cell Stem Cell. 2019;25(1):137–e148136. Winkler R, Gillis E, Lasman L, Safra M, Geula S, Soyris C, Nachshon A, Tai-Schmiedel J, Friedman N, Le-Trilling VTK, et al. Publisher Correction: m6A modification controls the innate immune response to infection by targeting type I interferons. Nat Immunol. 2019;20(2):243. Mishra T, Phillips S, Wu L. Method for the Enrichment of N(6)-Methyladenosine-Modified Cellular and HIV-1 RNA. Methods Mol Biol. 2024;2807:195–208. Asgari MM, Ray GT, Quesenberry CP Jr., Katz KA, Silverberg MJ. Association of Multiple Primary Skin Cancers With Human Immunodeficiency Virus Infection, CD4 Count, and Viral Load. JAMA Dermatol. 2017;153(9):892–6. Nicolau IA, Moineddin R, Brooks JD, Antoniou T, Gillis JL, Kendall CE, Cooper C, Cotterchio M, Salters K, Smieja M, et al. Associations of CD4 Cell Count Measures With Infection-Related and Infection-Unrelated Cancer Risk Among People With HIV. J Acquir Immune Defic Syndr. 2024;96(5):447–56. Cai S, Zhou J, Luo X, Zhang C, Jin S, Ren J, Cui J. Phase transition of WTAP regulates m6A modification of interferon-stimulated genes. eLife 2025. Han X, Zhu Y, Ke J, Zhai Y, Huang M, Zhang X, He H, Zhang X, Zhao X, Guo K, et al. Progression of m(6)A in the tumor microenvironment: hypoxia, immune and metabolic reprogramming. Cell Death Discov. 2024;10(1):331. Comoglio PM, Trusolino L, Boccaccio C. Known and novel roles of the MET oncogene in cancer: a coherent approach to targeted therapy. Nat Rev Cancer. 2018;18(6):341–58. Dai L, Trillo-Tinoco J, Cao Y, Bonstaff K, Doyle L, Del Valle L, Whitby D, Parsons C, Reiss K, Zabaleta J, et al. Targeting HGF/c-MET induces cell cycle arrest, DNA damage, and apoptosis for primary effusion lymphoma. Blood. 2015;126(26):2821–31. He WQ, Pang W, Li N, Li AQ, Li YH, Lu Y, Shen F, Xin R, Song TZ, Tian RR, et al. IFI27 inhibits HIV-1 replication by degrading Gag protein through the ubiquitin-proteasome pathway. J Virol. 2024;98(11):e0135624. Huang S, Zhao J, Song J, Li Y, Zuo R, Sa Y, Ma Z, OuYang H. Interferon alpha-inducible protein 27 (IFI27) is a prognostic marker for pancreatic cancer based on comprehensive bioinformatics analysis. Bioengineered. 2021;12(1):8515–28. Schoggins JW, Wilson SJ, Panis M, Murphy MY, Jones CT, Bieniasz P, Rice CM. A diverse range of gene products are effectors of the type I interferon antiviral response. Nature. 2011;472(7344):481–5. Zicari S, Sessa L, Cotugno N, Ruggiero A, Morrocchi E, Concato C, Rocca S, Zangari P, Manno EC, Palma P. Immune Activation, Inflammation, and Non-AIDS Co-Morbidities in HIV-Infected Patients under Long-Term ART. Viruses 2019, 11(3). Zhang C, Chen J, Ren J, Li X, Zhang Y, Huang B, Xu Y, Dong L, Cao Y. N(6)-methyladenosine levels in peripheral blood RNA: a potential diagnostic biomarker for colorectal cancer. Cancer Cell Int. 2024;24(1):96. Tirumuru N, Wu L. HIV-1 envelope proteins up-regulate N(6)-methyladenosine levels of cellular RNA independently of viral replication. J Biol Chem. 2019;294(9):3249–60. Rinaldi S, Pallikkuth S, Cameron M, de Armas LR, Cotugno N, Dinh V, Pahwa R, Richardson B, Saini SR, Rocca S, et al. Impact of Early Antiretroviral Therapy Initiation on HIV-Specific CD4 and CD8 T Cell Function in Perinatally Infected Children. J Immunol. 2020;204(3):540–9. Franceschi S, Lise M, Clifford GM, Rickenbach M, Levi F, Maspoli M, Bouchardy C, Dehler S, Jundt G, Ess S, et al. Changing patterns of cancer incidence in the early- and late-HAART periods: the Swiss HIV Cohort Study. Br J Cancer. 2010;103(3):416–22. Ping XL, Sun BF, Wang L, Xiao W, Yang X, Wang WJ, Adhikari S, Shi Y, Lv Y, Chen YS, et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 2014;24(2):177–89. Du Y, Hou G, Zhang H, Dou J, He J, Guo Y, Li L, Chen R, Wang Y, Deng R, et al. SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function. Nucleic Acids Res. 2018;46(10):5195–208. Yu F, Wei J, Cui X, Yu C, Ni W, Bungert J, Wu L, He C, Qian Z. Post-translational modification of RNA m6A demethylase ALKBH5 regulates ROS-induced DNA damage response. Nucleic Acids Res. 2021;49(10):5779–97. Dai XY, Shi L, Li Z, Yang HY, Wei JF, Ding Q. Main N6-Methyladenosine Readers: YTH Family Proteins in Cancers. Front Oncol. 2021;11:635329. Mackelprang RD, Filali-Mouhim A, Richardson B, Lefebvre F, Katabira E, Ronald A, Gray G, Cohen KW, Klatt NR, Pecor T, et al. Upregulation of IFN-stimulated genes persists beyond the transitory broad immunologic changes of acute HIV-1 infection. iScience. 2023;26(4):106454. Ji M, Ganesan S, Xia B, Huo Y. Targeting c-MET Alterations in Cancer: A Review of Genetic Drivers and Therapeutic Implications. Cancers (Basel) 2025, 17(9). Ataie-Kachoie P, Pourgholami MH, Morris DL. Inhibition of the IL-6 signaling pathway: a strategy to combat chronic inflammatory diseases and cancer. Cytokine Growth Factor Rev. 2013;24(2):163–73. Meissl K, Macho-Maschler S, Muller M, Strobl B. The good and the bad faces of STAT1 in solid tumours. Cytokine. 2017;89:12–20. Chen RH, Du Y, Han P, Wang HB, Liang FY, Feng GK, Zhou AJ, Cai MY, Zhong Q, Zeng MS, et al. ISG15 predicts poor prognosis and promotes cancer stem cell phenotype in nasopharyngeal carcinoma. Oncotarget. 2016;7(13):16910–22. Huang H, Lv J, Huang Y, Mo Z, Xu H, Huang Y, Yang L, Wu Z, Li H, Qin Y. IFI27 is a potential therapeutic target for HIV infection. Ann Med. 2022;54(1):314–25. McFadden MJ, McIntyre ABR, Mourelatos H, Abell NS, Gokhale NS, Ipas H, Xhemalce B, Mason CE, Horner SM. Post-transcriptional regulation of antiviral gene expression by N6-methyladenosine. Cell Rep. 2021;34(9):108798. Song H, Song J, Cheng M, Zheng M, Wang T, Tian S, Flavell RA, Zhu S, Li HB, Ding C, et al. METTL3-mediated m(6)A RNA methylation promotes the anti-tumour immunity of natural killer cells. Nat Commun. 2021;12(1):5522. Guirguis AA, Ofir-Rosenfeld Y, Knezevic K, Blackaby W, Hardick D, Chan YC, Motazedian A, Gillespie A, Vassiliadis D, Lam EYN, et al. Inhibition of METTL3 Results in a Cell-Intrinsic Interferon Response That Enhances Antitumor Immunity. Cancer Discov. 2023;13(10):2228–47. Carmona-Perez L, Dagenais-Lussier X, Mai LT, Stogerer T, Swaminathan S, Isnard S, Rice MR, Barnes BJ, Routy JP, van Grevenynghe J et al. The TLR7/IRF-5 axis sensitizes memory CD4 + T cells to Fas-mediated apoptosis during HIV-1 infection. JCI Insight 2023, 8(13). Urban-Wojciuk Z, Khan MM, Oyler BL, Fahraeus R, Marek-Trzonkowska N, Nita-Lazar A, Hupp TR, Goodlett DR. The Role of TLRs in Anti-cancer Immunity and Tumor Rejection. Front Immunol. 2019;10:2388. Additional Declarations No competing interests reported. Supplementary Files MishraTables12updated111225.docx Cite Share Download PDF Status: Published Journal Publication published 21 Mar, 2026 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Revision requested 05 Feb, 2026 Reviews received at journal 03 Feb, 2026 Reviewers agreed at journal 25 Jan, 2026 Reviews received at journal 29 Nov, 2025 Reviewers agreed at journal 16 Nov, 2025 Reviewers agreed at journal 16 Nov, 2025 Reviewers invited by journal 14 Nov, 2025 Editor assigned by journal 14 Nov, 2025 Editor invited by journal 13 Nov, 2025 Submission checks completed at journal 12 Nov, 2025 First submitted to journal 12 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":244207,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStatistical analyses of three measurable parameters between PLWH with or without cancer. (A)\u003c/strong\u003e age, \u003cstrong\u003e(B) \u003c/strong\u003eHIV-1 viral load (RNA copies/mL) in non-suppressed PLWH, and \u003cstrong\u003e(C) \u003c/strong\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cell counts/mm\u003csup\u003e3\u003c/sup\u003e blood. \u003cstrong\u003e(A-C) \u003c/strong\u003eMann-Whitney U test was used for the statistical analyses. \u003cem\u003ep\u003c/em\u003e values are labeled on the figures.\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8070380/v1/4a1d36e4b9645d06d39d2523.jpeg"},{"id":100151854,"identity":"f35eb5f9-a268-492a-b19e-90a6f6a08883","added_by":"auto","created_at":"2026-01-13 13:34:22","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of RNA m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA levels in PBMCs from PLWH with or without cancer.\u003c/strong\u003e Total cellular RNA isolated from PBMCs of 43 PLWH with (n = 15) or without cancer (n = 28) were subjected to m\u003csup\u003e6\u003c/sup\u003eA ELISA (200 ng of RNA/sample). The box plot represents the mean of absolute m\u003csup\u003e6\u003c/sup\u003eA amounts in cancer (+) and cancer (-) PLWH. Non-parametric unpaired Mann Whitney U-test was performed to estimate levels of significance (** \u003cem\u003ep\u003c/em\u003e ≤ 0.01).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8070380/v1/6ae5b718553d8045e89134db.jpeg"},{"id":100379851,"identity":"5a7a1620-6813-4001-8097-bf08cccdfd47","added_by":"auto","created_at":"2026-01-16 09:45:01","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":373045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelations of m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA RNA levels from PBMCs of PLWH with HIV-1 RNA copy numbers and CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cell counts.\u003c/strong\u003e Simple linear regression analyses of cellular RNA m\u003csup\u003e6\u003c/sup\u003eA amounts and plasma HIV-1 RNA copies/mL (A, B and C) or blood CD4\u003csup\u003e+\u003c/sup\u003e T cell counts (D, E and F). RNA samples were from PBMCs of \u003cstrong\u003e(A, D) \u003c/strong\u003eall PLWH (n = 43), \u003cstrong\u003e(B, E)\u003c/strong\u003e PLWH without cancer (n = 28), and \u003cstrong\u003e(C, F)\u003c/strong\u003e PLWH with cancer (n =15). Spearman correlation test was used to assess the relationship between the measured parameters. The correlation coefficient and \u003cem\u003ep\u003c/em\u003e values for each comparison are labeled over each graph\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8070380/v1/c28e0b6fbc3c535c89acd788.jpeg"},{"id":100151858,"identity":"6a2694c1-1afc-4628-8ce1-e0b56dc5a3b5","added_by":"auto","created_at":"2026-01-13 13:34:23","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":309242,"visible":true,"origin":"","legend":"\u003cp\u003eRelative mRNA expression levels of m\u003csup\u003e6\u003c/sup\u003eA regulators in PBMCs from PLWH. RT-qPCR was performed using RNA of PBMCs from the PLWH with cancer (n = 15) or without cancer (n = 28). Relative expression levels were determined by normalizing to \u003cem\u003eGAPDH\u003c/em\u003e and setting the average expression level in cancer-negative PLWH to 1. Relative mRNA expression levels of \u003cstrong\u003e(A)\u003c/strong\u003e m\u003csup\u003e6\u003c/sup\u003eA writer complex genes, \u003cstrong\u003e(B)\u003c/strong\u003e m\u003csup\u003e6\u003c/sup\u003eA eraser genes, and \u003cstrong\u003e(C)\u003c/strong\u003e m\u003csup\u003e6\u003c/sup\u003eA reader genes. Non-parametric unpaired Mann Whitney U-test was used to estimate the levels of significance (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e ≤ 0.01, ***\u003cem\u003ep \u003c/em\u003e≤ 0.001, ****\u003cem\u003ep \u003c/em\u003e≤0.0001, ns, not significant).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8070380/v1/7df57272aa0056c006c160b0.jpeg"},{"id":100367942,"identity":"b478d78c-8257-4fd9-aa4e-3ca552270227","added_by":"auto","created_at":"2026-01-16 07:57:28","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":564237,"visible":true,"origin":"","legend":"\u003cp\u003eCancer differentially regulates mRNA expression of IFN-I-responsive genes in PLWH. Relative gene expression was measured in PBMCs from PLWH with cancer (n = 15) or without cancer (n = 28). \u003cstrong\u003e(A)\u003c/strong\u003e Plate map depicting gene symbols for 84 IFN-I-responsive genes. \u003cstrong\u003e(B)\u003c/strong\u003e Heat maps show the average differential gene expression in PBMCs of cancer-positive compared with cancer-negative PLWH. The red and blue colors represent upregulated and downregulated genes, respectively. The scales of fold changes are shown next to the legends. Data were analyzed utilizing the web-based RT\u003csup\u003e2\u003c/sup\u003e profiler PCR array data analysis program (https://geneglobe.qiagen.com/us/analyze).\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8070380/v1/91f7a4615886d04e2b5afe1e.jpeg"},{"id":100151873,"identity":"2a73a479-b12d-43a4-a015-76de4a124901","added_by":"auto","created_at":"2026-01-13 13:34:23","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":336479,"visible":true,"origin":"","legend":"\u003cp\u003eSignificantly regulated IFN-I-responsive genes in PBMCs from cancer-positive (+) compared with cancer-negative (-) PLWH. Relative gene expression levels were calculated from the RT\u003csup\u003e2\u003c/sup\u003e profiler PCR array as shown in Figure 5A. \u003cstrong\u003e(A)\u003c/strong\u003e Relative mRNA levels of top ten genes (based on fold change) significantly upregulated in PBMCs from cancer (+) relative to cancer (-) PLWH. \u003cstrong\u003e(B)\u003c/strong\u003e Three genes significantly downregulated in PBMCs from cancer (+) relative to cancer (-) PLWH. \u003cstrong\u003e(A \u003c/strong\u003eand\u003cstrong\u003e B)\u003c/strong\u003e Non-parametric unpaired Mann Whitney U-test was used to estimate levels of significance (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e≤ 0.01, ***\u003cem\u003ep\u003c/em\u003e ≤ 0.001, and ****\u003cem\u003ep\u003c/em\u003e ≤ 0.0001).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8070380/v1/681eb52014d87f2a24b58c10.jpeg"},{"id":105224376,"identity":"cf7f98ba-7183-4b43-ad91-8610a172bff5","added_by":"auto","created_at":"2026-03-23 16:14:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3200745,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8070380/v1/abb8bc6a-459d-415d-adb4-153840fecba6.pdf"},{"id":100151856,"identity":"9b02d5b1-246c-4182-8d8c-eae38d69b6a0","added_by":"auto","created_at":"2026-01-13 13:34:23","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":30004,"visible":true,"origin":"","legend":"","description":"","filename":"MishraTables12updated111225.docx","url":"https://assets-eu.researchsquare.com/files/rs-8070380/v1/4a627142fb05ebbad2069ca1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Elevated m 6 A RNA Modifications Associate with Immune Dysregulation and Cancer in People with HIV-1","fulltext":[{"header":"Background","content":"\u003cp\u003eThe eukaryotic RNAs have more than 170 types of modifications, among which \u003cem\u003eN\u003c/em\u003e\u003csup\u003e6\u003c/sup\u003e-methyladenosine (m\u003csup\u003e6\u003c/sup\u003eA) is the most abundant internal modification in mRNA and various non-coding RNAs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. m\u003csup\u003e6\u003c/sup\u003eA modification influence RNA metabolism by regulating splicing, nuclear export, translation efficiency and transcript stability [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The dynamic m\u003csup\u003e6\u003c/sup\u003eA modifications are installed by writer enzymes [methyltransferase like 3/14 (METTL3/14) and Wilms tumor 1-associated protein (WTAP)], removed by erasers [AlkB homolog 5 (ALKBH5) and fat mass and obesity-associated protein (FTO)], and interpreted by reader proteins [YTH domain-containing family 1\u0026ndash;3 (YTHDF1-3), collectively called as m\u003csup\u003e6\u003c/sup\u003eA regulatory machinery [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The dysregulation of RNA m\u003csup\u003e6\u003c/sup\u003eA modification and its regulatory machinery, including writers, erasers and readers are frequently observed in pathogenesis of various diseases including viral infections and cancers [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. m\u003csup\u003e6\u003c/sup\u003eA modifications have been found on both HIV-1and host transcripts, depicting an intricate role for epitranscriptomic regulation in viral replication, immune evasion, and latency reactivation [\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. HIV-1 not only exploits the host m\u003csup\u003e6\u003c/sup\u003eA methylation machinery to enhance its gene expression and replication, but also modulates the expression of m\u003csup\u003e6\u003c/sup\u003eA regulators, leading to broader effects on host immune pathways and gene expression [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur previous study showed that combined antiretroviral therapy (ART) significantly suppresses RNA m\u003csup\u003e6\u003c/sup\u003eA levels in peripheral blood mononuclear cells (PBMCs) from people living with HIV-1 (PLWH) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Despite the success of ART, PLWH exhibit a significantly increased risk of developing certain cancers, including Kaposi\u0026rsquo;s sarcoma, non-Hodgkin lymphoma, and cervical cancers [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This increased cancer risk is partially attributed to chronic immune activation, persistent inflammation, co-infections with oncogenic viruses, and impaired immune surveillance [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the contribution of RNA modifications, especially m\u003csup\u003e6\u003c/sup\u003eA methylation to oncogenesis in PLWH remains underexplored.\u003c/p\u003e\u003cp\u003ePrevious studies have shown the oncogenic roles of several m\u003csup\u003e6\u003c/sup\u003eA regulatory proteins, such as METTL3, METTL14, FTO and YTHDF2 proteins, in promoting proliferation, metastasis and resistance to apoptosis across various cancer types [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Given the dual role of m\u003csup\u003e6\u003c/sup\u003eA in modulating antiviral immunity and cancer-associated gene expression, it is plausible that aberrant m\u003csup\u003e6\u003c/sup\u003eA signaling may serve as a mechanistic bridge between chronic HIV-1 infection and cancer development. Notably, m\u003csup\u003e6\u003c/sup\u003eA also regulates the expression and function of interferon-stimulated genes (ISGs), which are central to antiviral defense but can paradoxically promote tumorigenesis under chronic activation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This intersection raises the possibility that chronic HIV-1-associated immune activation, combined with epitranscriptomic dysregulation, may influence cancer susceptibility through sustained interferon (IFN) signaling and ISG modulation.\u003c/p\u003e\u003cp\u003eIn this study, we investigated the m\u003csup\u003e6\u003c/sup\u003eA RNA methylations in PBMCs from 43 de-identified aging PLWH with or without cancer at average age of 56 and 60, respectively. We found significantly elevated RNA m\u003csup\u003e6\u003c/sup\u003eA levels in PLWH with cancer, which were accompanied by upregulated expression of mRNA encoding the m\u003csup\u003e6\u003c/sup\u003eA writer complex and reader YTHDF1-3 proteins. Furthermore, we observed that the cellular RNA m\u003csup\u003e6\u003c/sup\u003eA levels correlated with higher HIV-1 RNA copy numbers and lower CD4\u003csup\u003e+\u003c/sup\u003e T cell counts. Importantly, we demonstrated that cancer development in PLWH was associated with aberrant expression of IFN-I-responsive genes, suggesting a link between m\u003csup\u003e6\u003c/sup\u003eA-modulated immune responses and oncogenesis. These findings provide new insights into how m\u003csup\u003e6\u003c/sup\u003eA RNA modifications may contribute to the dysregulation of innate immunity and the development of HIV-1-associated cancers.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy participants and de-identified PBMCs samples\u003c/h2\u003e\u003cp\u003ePBMCs were obtained from a total of 43 PLWH with cancer (n\u0026thinsp;=\u0026thinsp;15) or without cancer (n\u0026thinsp;=\u0026thinsp;28) and de-identified sample details were described in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, respectively. Thirteen of the de-identified samples (n\u0026thinsp;=\u0026thinsp;13) were provided by the AIDS and Cancer Specimen Resource funded by the National Cancer Institute, NIH. Individuals (n\u0026thinsp;=\u0026thinsp;30) attending the University of Iowa Virology Clinic were invited to participate in this study and, following written informed consent, provided blood samples. The informed consent to participate was obtained from all of the participants in the study. PMBCs were collected from PLWH with a documented history of cancer, or from those without cancer. Blood samples were obtained for the preparation of PBMCs as described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Medical records of those with HIV-1 were reviewed. CD4\u003csup\u003e+\u003c/sup\u003e T cells count was either obtained at the same visit, or the most recent (within 2 years) in those with over 2 years viral load suppression, and HIV-1 viral load were documented. Plasma HIV-1 viral load was conducted using the COBAS\u0026reg; AmpliPrep/COBAS\u0026reg; TaqMan HIV-1 test (Roche). PBMCs were preserved in liquid nitrogen until use. Statistical analyses of the age, HIV-1 viral load, and CD4\u003csup\u003e+\u003c/sup\u003e T cells counts between the cancer and non-cancer groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDetails of PBMC samples from de-identified PLWH with cancer\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"14\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParticipant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHIV-1 viral load (RNA copies/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCD4\u0026thinsp;+\u0026thinsp;T cell count/mm\u003csup\u003e3\u003c/sup\u003e blood\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCancer type \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCancer stage\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eTreatment stage and type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eART regimen\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eDrugs of abuse\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eMarijuana use\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e115,037\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKS \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHigh Risk\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e67,633\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e236\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLow Risk\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e99,139\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e195\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLow Risk\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54,766\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHigh Risk\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17,078\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHigh Risk\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e248,068\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHigh Risk\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e223\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLow Risk\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eART\u0026thinsp;~\u0026thinsp;2.5 mos prior to sample collection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003elopinavir/ritonavir and emtricitabine/\u003c/p\u003e\u003cp\u003etenofovir\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e148\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e606\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eProstate cancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRemission\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eProstatectomy 2006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF \u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS \u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1,497\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHodgkin\u0026rsquo;s disease\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRemission\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSplenectomy, 1980s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eYes, many\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e885\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNon-melanocytic skin cancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCured\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSurgical removal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e262\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRemission\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eDiagnosed 2012-\u003c/p\u003e\u003cp\u003eresponded to ART\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e139\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e506\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLarge B cell lymphoma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRemission\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eTreated with RCHOP 2023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e941\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eVulvar cancer, Anal squamous cell x 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eremission, active\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eResected 2022, lymph node resection, XRT, Resected 2024\u0026ndash;2025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eJuluca\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e703\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAnal dysplasia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRemoved\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eResected 2016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11,500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e718\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLung cancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eResected\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eAdenocarcinoma, resected and XRT 2022\u0026ndash;2023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eYes, remote\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e56\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e55,813\u0026thinsp;\u0026plusmn;\u0026thinsp;72,821\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e460\u0026thinsp;\u0026plusmn;\u0026thinsp;414\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c13\" namest=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"14\"\u003eOnly de-identified information is shown in the table. The informed consent to participate was obtained from all of the participants.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"14\"\u003e\u003csup\u003ea\u003c/sup\u003e Cancer present or being treated at time of blood acquisition. \u003csup\u003eb\u003c/sup\u003e Samples from participants 1\u0026ndash;7 were provided by the AIDS and Cancer Specimen Resource funded by the National Cancer Institute, NIH; Cancer stage is based on ACTG Staging for KS using tumor extent, immune status, and systemic illness factors. \u003csup\u003ec\u003c/sup\u003e M: male. \u003csup\u003ed\u003c/sup\u003e KS: Kaposi sarcoma. \u003csup\u003ee\u003c/sup\u003e Samples from participants 8\u0026ndash;15 were provided by the University of Iowa HIV Clinic. \u003csup\u003ef\u003c/sup\u003e F: female. \u003csup\u003eg\u003c/sup\u003e S: HIV-1 RNA suppressed (viral load\u0026thinsp;\u0026le;\u0026thinsp;20 copies/mL).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDetails of PBMC samples from de-identified PLWH without cancer\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParticipant ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHIV-1 viral load (RNA copies/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCD4\u0026thinsp;+\u0026thinsp;T cell count/mm\u003csup\u003e3\u003c/sup\u003e blood\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eART regimen\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eDrugs of abuse\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMarijuana use\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e511\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLamivudine/Zidovudine/Nevirapine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60,785\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e154\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e428\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLamivudine/Zidovudine/Nevirapine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e485\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLamivudine/Zidovudine/Efavirenz\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e66,714\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e337\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e112,030\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e211\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e661\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eYes - many\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e338\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGenvoya\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1,400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDovato\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e267\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eJuluca\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e749\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTriumeq\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e790\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDescovy\u0026thinsp;+\u0026thinsp;Tivicay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1,667\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCabenuva\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e835\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDovato\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e133\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1,059\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDovato\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1,078\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e338\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGenvoya\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e604\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e846\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31,500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e147\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e105\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e235\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e634\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDescovy\u0026thinsp;+\u0026thinsp;Tivicay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e832\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e620\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e460\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eOdefsey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1,343\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e296\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBiktarvy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e60\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e17,004\u0026thinsp;\u0026plusmn;\u0026thinsp;32,656\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e619\u0026thinsp;\u0026plusmn;\u0026thinsp;399\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eOnly de-identified information is shown in the table. The informed consent to participate was obtained from all of the participants.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003ea\u003c/sup\u003e Samples from participants 1\u0026ndash;6 were provided by the AIDS and Cancer Specimen Resource funded by the National Cancer Institute, NIH. \u003csup\u003eb\u003c/sup\u003e M: male. \u003csup\u003ec\u003c/sup\u003e F: female. \u003csup\u003ed\u003c/sup\u003e Samples from participants 7\u0026ndash;28 were provided by the University of Iowa HIV Clinic. \u003csup\u003ee\u003c/sup\u003e S: HIV-1 RNA suppressed (viral load\u0026thinsp;\u0026le;\u0026thinsp;20 copies/mL).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCellular RNA isolation and RT-qPCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA isolation was performed using TRIzolT\u003csup\u003eM\u003c/sup\u003e reagent (ThermoFisher Scientific, 15596018) following manufacturers guidelines. The isolated RNA was quantified using a NanoDrop OneC spectrophotometer (ThermoFisher Scientific). DNase-treated RNA (500 ng) served as a template for cDNA synthesis, using the iScript cDNA Synthesis Kit following the manufacturer\u0026rsquo;s protocol (Bio-Rad, 1708890). qRT-PCR was performed with the iTaqSYBR Green PCR Kit (#1725124, Biorad) in the BioRad CFX96 Real-Time PCR system to evaluate the relative mRNA expression of the m\u003csup\u003e6\u003c/sup\u003eA writer complex genes (\u003cem\u003eMETTL3\u003c/em\u003e, \u003cem\u003eMETTL14\u003c/em\u003e, \u003cem\u003eWTAP\u003c/em\u003e, \u003cem\u003eRBM15\u003c/em\u003e, and \u003cem\u003eVIRMA\u003c/em\u003e), erasers (\u003cem\u003eALKBH5\u003c/em\u003e and \u003cem\u003eFTO\u003c/em\u003e), and readers (\u003cem\u003eYTHDF1-3\u003c/em\u003e) in PBMCs obtained from PLWH with cancer and without cancer. All reactions were performed in triplicate and normalized with \u003cem\u003eGAPDH\u003c/em\u003e as a housekeeping gene. The relative gene expression of each sample was calculated using the 2\u003csup\u003e\u0026minus;ΔΔ\u003cem\u003eC\u003c/em\u003et\u003c/sup\u003e formula. The primers for qRT-PCR were described in our previous study [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eRNA mA quantification by ELISA\u003c/h3\u003e\n\u003cp\u003eRNA m\u003csup\u003e6\u003c/sup\u003eA ELISA was performed according to the published protocol [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Briefly, 200 ng of total RNA per sample was quantified in triplicate. The primary m\u003csup\u003e6\u003c/sup\u003eA antibody solution (ABclonal, A19841) was prepared by diluting 1:10,000 in PBST (PBS with 0.1% Tween 20), and the secondary anti-rabbit IgG solution (Promega, W4011) was prepared by diluting 1:5000 in PBST. Each well was sequentially incubated with 100 \u0026micro;L of primary antibody solution, followed by 100 \u0026micro;L of secondary antibody solution. The signal was developed using 100 \u0026micro;L of 3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine substrate (ThermoFisher Scientific, 34021) and quenched with 100 \u0026micro;L of stop solution (2% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e). Absorbance was measured at 450 nm, and m\u003csup\u003e6\u003c/sup\u003eA levels were quantified relative to a standard curve generated using defined concentrations of m\u003csup\u003e6\u003c/sup\u003eA-modified RNA (EpiGentek, P-9005-96-PC).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eElevated RNA m\u003csup\u003e6\u003c/sup\u003eA levels in PBMCs from de-identified PLWH with cancer\u003c/h2\u003e\u003cp\u003eTo examine the potential effects of HIV-1 infection and cancer status on cellular RNA m\u003csup\u003e6\u003c/sup\u003eA levels \u003cem\u003eex vivo\u003c/em\u003e, we obtained PBMCs from 43 de-identified PLWH, including those with various types of cancer (n\u0026thinsp;=\u0026thinsp;15) and without cancer (n\u0026thinsp;=\u0026thinsp;28). The informed consent to participate was obtained from all of the participants in the study. The age, gender, HIV-1 plasma viral load, and CD4\u003csup\u003e+\u003c/sup\u003e T cell counts, cancer type, treatment stage and type, ART regimen, drugs of abuse, use of marijuana of de-identified PLWH with cancer are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The age, gender, HIV-1 plasma viral load, CD4\u003csup\u003e+\u003c/sup\u003e T cell counts, ART regimen, drug of abuse, and use of marijuana of de-identified PLWH without cancer are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Only de-identified information is provided in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Statistical analyses indicated that HIV-1 viral load was higher in participants in the cancer group compared to those in the non-cancer groups, while the age and CD4\u003csup\u003e+\u003c/sup\u003e T cell counts did not show statistical differences between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRelative m\u003csup\u003e6\u003c/sup\u003eA levels in RNA from PBMCs of PLWH were quantified using m\u003csup\u003e6\u003c/sup\u003eA ELISA [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. PLWH with cancer exhibited approximately 2.8-fold higher m\u003csup\u003e6\u003c/sup\u003eA levels than those without cancer (p\u0026thinsp;\u0026le;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The increased m\u003csup\u003e6\u003c/sup\u003eA levels are also correlated with higher viral load in the cancer group compared to non-cancer group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This observation extends our previous findings that viremic individuals show elevated m\u003csup\u003e6\u003c/sup\u003eA levels compared to PLWH on ART [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These findings suggest a potential link between HIV-1 infection and elevated m\u003csup\u003e6\u003c/sup\u003eA levels, which may contribute to dysregulation of host gene expression and possibly increase the risk of cancer development in PLWH.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCorrelation of cellular RNA mA levels with HIV-1 viral load and CD4 T cell counts\u003c/h3\u003e\n\u003cp\u003eTo determine whether RNA m\u003csup\u003e6\u003c/sup\u003eA levels are associated with HIV-1 replication, we analyzed the correlation between m\u003csup\u003e6\u003c/sup\u003eA levels in PBMCs and plasma HIV-1 RNA copies of PLWH. In the combined dataset (n\u0026thinsp;=\u0026thinsp;43), cellular m\u003csup\u003e6\u003c/sup\u003eA RNA levels showed a significant positive correlation with HIV-1 RNA levels (p\u0026thinsp;=\u0026thinsp;0.0005; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). When analyzed separately, a significant negative correlation was observed between m\u003csup\u003e6\u003c/sup\u003eA levels and HIV-1 RNA copies in PLWH without cancer (n\u0026thinsp;=\u0026thinsp;28, p\u0026thinsp;=\u0026thinsp;0.0082; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), whereas a strong positive correlation was found in PLWH with cancer (n\u0026thinsp;=\u0026thinsp;15, p\u0026thinsp;=\u0026thinsp;0.0059; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These data demonstrate that elevated HIV-1 viral load and cancer tumor microenvironment is associated with increased RNA m\u003csup\u003e6\u003c/sup\u003eA levels in PBMCs, suggesting that HIV-1 viral load and tumor microenvironment in PLWH with cancer enhance cellular RNA m\u003csup\u003e6\u003c/sup\u003eA modifications.\u003c/p\u003e\u003cp\u003eWe also assessed the association between cellular m\u003csup\u003e6\u003c/sup\u003eA RNA levels and CD4\u003csup\u003e+\u003c/sup\u003e T cell counts of PLWH. In the overall cohort, a significant negative correlation was observed (n\u0026thinsp;=\u0026thinsp;43, p\u0026thinsp;=\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This trend remained significant among PLWH with cancer (n\u0026thinsp;=\u0026thinsp;15, p\u0026thinsp;=\u0026thinsp;0.02; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), but not in PLWH without cancer (n\u0026thinsp;=\u0026thinsp;28, p\u0026thinsp;=\u0026thinsp;0.2725; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The increased m\u003csup\u003e6\u003c/sup\u003eA levels and the association with CD4\u003csup\u003e+\u003c/sup\u003e T cell decline in PLWH with cancer may also contribute to heightened cancer risk in PLWH [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eExpression of m\u003csup\u003e6\u003c/sup\u003eA regulators genes in PBMCs from PLWH with or without cancer\u003c/h2\u003e\u003cp\u003eTo determine whether elevated m\u003csup\u003e6\u003c/sup\u003eA RNA levels in PLWH with cancer are driven by altered expression of m\u003csup\u003e6\u003c/sup\u003eA regulatory genes, we measured the mRNA levels of major genes encoding m\u003csup\u003e6\u003c/sup\u003eA writers, erasers, and readers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We observed a significant increase in the expression of the core methyltransferase components METTL3, METTL14, as well as the accessory proteins RBM15 and VIRMA, in PBMCs from PLWH with cancer compared to those without cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, WTAP, an essential scaffolding subunit of the methyltransferase complex, showed no change in expression, suggesting that its regulatory function may be modulated post-transcriptionally or through other mechanisms [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In the m\u003csup\u003e6\u003c/sup\u003eA erasers, we detected a modest increase in \u003cem\u003eALKBH5\u003c/em\u003e mRNA expression, while \u003cem\u003eFTO\u003c/em\u003e mRNA expression remained unchanged according to statistical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The mRNA levels of the m\u003csup\u003e6\u003c/sup\u003eA reader proteins YTHDF1-3 were significantly elevated in PLWH with cancer compared to those without cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These findings suggest that the increased m\u003csup\u003e6\u003c/sup\u003eA RNA methylation levels may be driven by transcriptional upregulation of m\u003csup\u003e6\u003c/sup\u003eA regulatory genes. However, it is important to note that mRNA expression levels do not always correspond to protein abundance or enzymatic activity, and further studies are needed to assess protein-level changes and post-translational regulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eDysregulated expression of IFN-I-responsive genes in PLWH with cancer\u003c/h2\u003e\u003cp\u003eGiven the functional significance of m\u003csup\u003e6\u003c/sup\u003eA modification and importance of IFN-I-associated genes in regulating both HIV-1 infection and cancer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], we explored the expression of IFN-I-responsive genes in PLWH with cancer compared to individuals without cancer. Using an RT-qPCR array we analyzed the mRNA levels of 84 IFN-I-responsive genes, including interferons, signaling molecules, receptors, ISGs, and IFN-resistance markers. The assay allowed us to estimate the RNA levels of 84 IFN-I associated genes in one plate with respect to five housekeeping genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Heat maps were generated to depict the average change in gene expression in PLWH with cancer (n\u0026thinsp;=\u0026thinsp;15) or without cancer (n\u0026thinsp;=\u0026thinsp;28). Overall, many IFN-I-responsive genes expression levels were upregulated in caner-positive with respect to cancer-negative PLWH (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSignificantly regulated IFN-I-responsive gene expression in PLWH with cancer\u003c/h2\u003e\u003cp\u003eUsing the IFN-I-responsive gene expression array, we identified a panel of genes significantly upregulated in PLWH with cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). A comparative analysis of the top ten upregulated genes (based on fold change) between cancer-positive and cancer-negative PLWH revealed a distinct transcriptional signature (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Among these, MET proto-oncogene, receptor tyrosine kinase (\u003cem\u003eMET\u003c/em\u003e) and IFN-alpha-inducible protein 27 (\u003cem\u003eIFI27\u003c/em\u003e) mRNA levels were the most significantly elevated in cancer-positive PLWH, exhibiting\u0026thinsp;~\u0026thinsp;32-fold (p\u0026thinsp;=\u0026thinsp;0.0001) and ~\u0026thinsp;14-fold (p\u0026thinsp;=\u0026thinsp;0.0001) increases, respectively. This observation is consistent with our prior findings that HIV-1 infection induces \u003cem\u003eMET\u003c/em\u003e and \u003cem\u003eIFI27\u003c/em\u003e expression in viremic PLWH compared to HIV-1-suppressed individuals on ART [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although the role of IFI27 in HIV-1 infection and progression have been reported [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], its function in context of oncogenesis in PLWH remains to be explored.\u003c/p\u003e\u003cp\u003eMET is involved in cell proliferation, survival, and metastasis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and its overexpression has been implicated in various cancers [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], suggesting that its upregulation in the HIV-1 and cancer context may contribute to tumorigenesis. IFI27, a well-known ISG, has been implicated in both viral infection responses [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and cancer progression through its regulation of apoptosis and immune evasion [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Several other IFN-I-responsive genes were also significantly upregulated in cancer-positive PLWH, including \u003cem\u003eISG15\u003c/em\u003e, C-X-C motif chemokine ligand 10 (\u003cem\u003eCXCL10\u003c/em\u003e), interferon-induced protein with tetratricopeptide repeats 3 (\u003cem\u003eIFIT3\u003c/em\u003e), interleukin 6 (\u003cem\u003eIL6\u003c/em\u003e), interferon regulatory factor 7 (\u003cem\u003eIRF7\u003c/em\u003e), interferon-induced transmembrane protein 3 (\u003cem\u003eIFITM3\u003c/em\u003e), interferon-gamma inducible protein 16 (\u003cem\u003eIFI16\u003c/em\u003e), and signal transducer and activator of transcription 1 (\u003cem\u003eSTAT1\u003c/em\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, three innate immunity regulatory genes were significantly downregulated (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in cancer-positive PLWH compared to cancer-negative PLWH, including genes encoding interferon regulatory factor 5 (\u003cem\u003eIRF5\u003c/em\u003e), Toll-like receptor 7 (\u003cem\u003eTLR7\u003c/em\u003e), and \u003cem\u003eTLR8\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These genes are known to contribute to chronic immune activation, inflammation, and tumor-promoting immune landscapes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Together, these data suggest a dynamic reprogramming of IFN-I signaling in PLWH with cancer, potentially driven by chronic infection and altered m\u003csup\u003e6\u003c/sup\u003eA-mediated gene regulation.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe epitranscriptomic m\u003csup\u003e6\u003c/sup\u003eA RNA modification is the most prevalent internal modification on eukaryotic mRNA and is dynamically regulated by a set of proteins known as m\u003csup\u003e6\u003c/sup\u003eA regulators controlling the fate of RNA. We found that RNA m\u003csup\u003e6\u003c/sup\u003eA levels of PBMCs from PLWH with cancer were higher compared to PBMCs from PLWH without cancer. This suggest that cancers further enhance m\u003csup\u003e6\u003c/sup\u003eA RNA modification in PLWH. These observations align with previous reports [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] that enhanced m\u003csup\u003e6\u003c/sup\u003eA modification levels can serve as a biomarker for both HIV-1 infection and cancer progression.\u003c/p\u003e\u003cp\u003eTo explore the connection between m\u003csup\u003e6\u003c/sup\u003eA RNA modification, HIV-1 replication, and cancer status, we performed correlation analysis. We observed a positive correlation between m\u003csup\u003e6\u003c/sup\u003eA levels and HIV-1 RNA copies when data from all PLWH were analyzed collectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), supporting previous findings that HIV-1 infection and treatment with recombinant HIV-1 gp120 enhances cellular RNA m\u003csup\u003e6\u003c/sup\u003eA levels [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Interestingly, when stratified by cancer status, a positive correlation between m\u003csup\u003e6\u003c/sup\u003eA and viral load was observed in PLWH with cancer, whereas a negative correlation was found in PLWH without cancer. The negative correlation may be influenced by lower average plasma HIV-1 RNA copies in the non-cancer group (mean\u0026thinsp;=\u0026thinsp;55,813 copies/mL) compared to cancer group (mean\u0026thinsp;=\u0026thinsp;17,004 copies/mL). The smaller sample size may skew the analysis outcome. Performing the correlation analysis on a large cohort can further support the findings.\u003c/p\u003e\u003cp\u003eWe observe a significant negative correlation in CD4\u003csup\u003e+\u003c/sup\u003e T cell counts and m\u003csup\u003e6\u003c/sup\u003eA RNA levels in combined dataset (n\u0026thinsp;=\u0026thinsp;43) and in cancer subgroup, while a non-significant trend was observed in non-cancer subgroup. Elevated m\u003csup\u003e6\u003c/sup\u003eA levels might be associated with reduced CD4⁺ T cell recovery in PLWH, particularly in those with cancer. Indeed, individuals with cancer had markedly lower average CD4⁺ counts (460\u0026thinsp;\u0026plusmn;\u0026thinsp;414) compared to those without cancer (619\u0026thinsp;\u0026plusmn;\u0026thinsp;399). These findings emphasize the importance of early HIV-1 diagnosis and regular monitoring of both CD4⁺ T cell counts and m\u003csup\u003e6\u003c/sup\u003eA RNA levels to assess cancer risk in PLWH [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOne limitation in our study is potential confounding variables in the cohort, such as HIV-1 viral load, CD4\u003csup\u003e+\u003c/sup\u003e T cell counts, ART adherence, and drug use. Our statistical analyses indicate that the difference in the age and CD4\u003csup\u003e+\u003c/sup\u003e T cell counts is not significant between the cancer and non-cancer groups, while the viral load is significantly higher in the cancer group compared to the non-cancer group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Our previous study showed significant higher RNA m\u003csup\u003e6\u003c/sup\u003eA levels in PBMCs from HIV-1 viremic individuals compared to those on ART [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, the higher viral load in the cancer group might contribute to the increased m\u003csup\u003e6\u003c/sup\u003eA RNA levels compared to the non-cancer group. Notably, in the cancer cohort, 6 out of 15 PLWH had not received ART, while in the non-cancer cohort, only 3 out of 28 PLWH had not undergone ART. Therefore, the higher viral load observed in the cancer group may be partly due to the larger proportion of individuals not receiving ART. However, we believe that our results are not solely explained by HIV-1 infection and ART status but are also influenced by the cancer status of the individuals.\u003c/p\u003e\u003cp\u003eBecause we observed elevated levels of m\u003csup\u003e6\u003c/sup\u003eA RNA modification in PLWH with cancer compared to PLWH without cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e), we hypothesized that this increase might be due to altered expression of m\u003csup\u003e6\u003c/sup\u003eA regulatory machinery, including writers, erasers, and readers. We thus analyzed the expression of key m\u003csup\u003e6\u003c/sup\u003eA regulatory genes. We observed a significant upregulation of METTL3 and METTL14 transcripts, the two catalytic components of the m\u003csup\u003e6\u003c/sup\u003eA methyltransferase complex, in cancer samples. Interestingly, mRNA expression of WTAP, a key regulatory subunit that stabilizes and scaffolds the methyltransferase complex [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This raises the possibility that post-transcriptional or post-translational modifications (PTMs) may regulate WTAP function in HIV-1-associated cancer, requires further studies. We also observed increased expression of RBM15 that guide m\u003csup\u003e6\u003c/sup\u003eA deposition at specific transcript regions. The upregulation of ALKBH5 mRNA expression was unexpected and suggests a more complex regulatory mechanism that might involve substrate specificity, cellular localization, or PTMs.\u003c/p\u003e\u003cp\u003eDue to the limited number of available cells, we could not assess protein expression levels in this study. However, our previous work indicated no significant differences in protein levels of these regulators between PLWH and healthy controls [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Thus, the observed increase in m\u003csup\u003e6\u003c/sup\u003eA levels in cancer samples cannot be solely attributed to RNA or protein abundance. It is possible that m\u003csup\u003e6\u003c/sup\u003eA regulatory enzyme activity is modulated by PTMs which are known to influence both writer and eraser functions [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Whether such PTM-based modulation contributes to elevated m\u003csup\u003e6\u003c/sup\u003eA levels in PLWH with cancer remains an open question and merits further investigation.\u003c/p\u003e\u003cp\u003eWe also found significant mRNA upregulation of \u003cem\u003eYTHDF1-3\u003c/em\u003e, which encode major members of the YTHDF family of m\u003csup\u003e6\u003c/sup\u003eA reader proteins, in samples from PLWH and cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These proteins bind m\u003csup\u003e6\u003c/sup\u003eA-modified transcripts and regulate their translation or decay. Previous studies have linked YTHDF proteins to tumorigenesis in various cancers, including hepatocellular carcinoma, leukemia, and glioblastoma [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. For instance, YTHDF2 promotes degradation of pro-apoptotic or differentiation-associated mRNAs, while, YTHDF1 enhances the translation of growth-promoting mRNAs and has been shown to correlate with poor prognosis in several malignancies. The elevated expression of YTHDF readers may reflect a cellular adaptation to chronic immune activation, viral persistence, or altered m\u003csup\u003e6\u003c/sup\u003eA dynamics induced by HIV-1 or ART. It remains to be determined whether these changes contribute functionally to tumor development or represent a compensatory mechanism in response to altered m\u003csup\u003e6\u003c/sup\u003eA dynamics.\u003c/p\u003e\u003cp\u003eTo further explore how m\u003csup\u003e6\u003c/sup\u003eA dysregulation intersects with immune signaling, we analyzed the expression profiles of 84 IFN-I-responsive genes and immune-related genes associated with HIV-1 pathogenesis and cancer progression. We found that mRNA levels of a subset of IFN-I responsive genes, including \u003cem\u003eMET, IFI27, ISG15, CXCL10, IFIT3, IL6, IRF7, IFITM3, IFI16\u003c/em\u003e, and \u003cem\u003eSTAT1\u003c/em\u003e, were significantly upregulated in cancer samples from PLWH (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). These genes and their encoded proteins play central roles in regulating antiviral immunity and inflammation. Of note, persistent expression of ISGs in PLWH, even under ART, suggests a state of chronic immune activation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eImportantly, many of these genes also have established roles in tumorigenesis. For instance, MET is a proto-oncogene implicated in tumor proliferation and metastasis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]; IL6 is a pro-inflammatory cytokine that supports tumor growth and immune evasion [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]; and STAT1, though typically antiviral, has context-dependent functions in promoting or suppressing tumorigenesis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Additionally, ISG15 and IFI27 modulate apoptosis and immune responses and are upregulated in various cancers and viral infections [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Notably, several of these transcripts such as \u003cem\u003eSTAT1, ISG15\u003c/em\u003e, and \u003cem\u003eCXCL10\u003c/em\u003e have been reported to be regulated by m\u003csup\u003e6\u003c/sup\u003eA modification [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This suggests that the m\u003csup\u003e6\u003c/sup\u003eA RNA modification may further shape the chronic inflammatory state observed in these PLWH. A recent study showed that inhibition of METTL3 stimulates IFN-I response by promoting double stranded RNA formation and promoting anti-tumor immunity [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConversely, we observed significant downregulation of IFN-I regulatory or pattern recognition receptor genes, such as IRF5, TLR7 and TLR8 in PLWH with cancer. These genes are vital for sensing viral RNA and activating IFN-I responses [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Their reduced expression may represent a mechanism of immune evasion or exhaustion in the tumor microenvironment, potentially contributing to impaired antiviral surveillance and increased cancer susceptibility in PLWH. Due to limited RNA availability from PBMCs in this study, we were unable to assess m\u003csup\u003e6\u003c/sup\u003eA methylation status of individual IFN-I-responsive transcripts. Collection of additional samples will be necessary to determine m\u003csup\u003e6\u003c/sup\u003eA levels and further elucidate its role in regulating RNA stability and translation in aging PLWH with cancer. Inflammation and immunosuppression induced by chronic virus infection and cancer can lead to a dysfunctional immune state [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. IFN-I-responsive transcripts might play an important role and exert their opposing effects during cancer progression and HIV-1 infection. This potential dual role needs further investigation in future studies.\u003c/p\u003e\u003cp\u003eWe believe that the interplay between HIV-1 infection, cancer, and interferon responses is highly complex and likely influenced by multiple, overlapping factors. It is difficult to conclusively determine whether the elevated IFN-I responses observed in our dataset are driven primarily by the presence of cancer, HIV-1 infection, or their combined effect. Importantly, our prior work has shown that the HIV-1 envelope protein gp120 can upregulate m\u003csup\u003e6\u003c/sup\u003eA RNA modification in host cells independently of active viral replication [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, IFN-I transcripts themselves are subject to m\u003csup\u003e6\u003c/sup\u003eA modification, which targets them for degradation and helps fine-tune immune homeostasis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, HIV-1 intron-containing RNAs have been shown to activate innate immune sensors and stimulate IFN-I responses. These studies support the possibility that HIV-1 infection can modulate both m\u003csup\u003e6\u003c/sup\u003eA dynamics and IFN-I responses through multiple mechanisms, independent of or in conjunction with cancer. Further functional studies are required to understand the complex mechanism undying the connection between HIV-1 infection, m\u003csup\u003e6\u003c/sup\u003eA RNA modification, and caner progression.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eCollectively, our findings suggest a complex immune landscape in HIV-1-associated cancers characterized by simultaneous overactivation of ISGs and suppression of innate sensing pathways. The interplay between m\u003csup\u003e6\u003c/sup\u003eA modifications and dysregulated interferon signaling may represent a critical axis in the development of HIV-1-associated cancer in aging populations.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHIV-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehuman immunodeficiency virus type 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePLWH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epeople living with HIV-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003em\u003csup\u003e6\u003c/sup\u003eA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eN\u003c/em\u003e\u003csup\u003e6\u003c/sup\u003e-methyladenosine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBMCs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eperipheral blood mononuclear cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eART\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eantiretroviral therapy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIFN-I\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etype I interferon\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eenzyme-linked immunosorbent assay\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ereverse transcription quantitative polymerase chain reaction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eISGs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003einterferon-stimulated genes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMETTL3/14\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emethyltransferase like 3/14\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eWTAP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eWilms tumor 1-associated protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eALKBH5\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAlkB homolog 5\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFTO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003efat mass and obesity-associated protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eYTHDF1-3\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eYTH domain-containing family 1\u0026ndash;3\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003e The study using human PBMCs from PLWH with and without cancer and consent to participate have been approved by the Institutional Review Board (IRB) of the University of Iowa. Individuals attending the University of Iowa Virology Clinic were invited to participate in this study and, following written informed consent, provided blood samples. The informed consent to participate was obtained from all of the participants in the study. The study was conducted according to the Declaration of Helsinki guidelines.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eConsent for publication has been obtained from the study participants through the approved IRB protocol as described above. Only de-identified information is shown in the sample summary Tables\u0026nbsp;1\u0026ndash;2.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the U.S. National Institutes of Health (NIH) grants P30CA086862 (supplement 24S1 and 25S1) and R61/R33AI169659 to L.W, and in part by the Veterans Administration Health Service grant BX000207 to J.T.S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: T.M., S.T., and L.W. Resources: J.T.S. and L.W. Methodology and Investigation: T.M. and S.T. Formal analysis: T.M., S.T., and L.W. Writing-Original Draft: T.M. and S.T., and L.W. Writing-Reviewing and Editing: J.S. and L.W. Visualization: T.M. and L.W. Supervision, Project administration, and Funding acquisition: L.W. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Ms. Bethany Wilms and Mr. Ashok Chaudhary for technical assistance and the Wu lab members for helpful discussion and suggestions. Thirteen de-identified specimens in this study (detailed in Tables 1-2 with de-identified information) were provided by the AIDS and Cancer Specimen Resource, funded by the National Cancer Institute, NIH.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this manuscript for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRoundtree IA, Evans ME, Pan T, He C. Dynamic RNA Modifications in Gene Expression Regulation. Cell. 2017;169(7):1187\u0026ndash;200.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao BS, Roundtree IA, He C. Post-transcriptional gene regulation by mRNA modifications. Nat Rev Mol Cell Biol. 2017;18(1):31\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeyer KD, Jaffrey SR. The dynamic epitranscriptome: N6-methyladenosine and gene expression control. Nat Rev Mol Cell Biol. 2014;15(5):313\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe PC, He C. m(6) A RNA methylation: from mechanisms to therapeutic potential. EMBO J. 2021;40(3):e105977.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarbieri I, Kouzarides T. Role of RNA modifications in cancer. Nat Rev Cancer. 2020;20(6):303\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Z, Liu F, Chen W, Liao Z, Zhang W, Zhang B, Liang H, Chu L, Zhang Z. The importance of N6-methyladenosine modification in tumor immunity and immunotherapy. Exp Hematol Oncol. 2022;11(1):30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePhillips S, Mishra T, Huang S, Wu L. Functional Impacts of Epitranscriptomic m(6)A Modification on HIV-1 Infection. Viruses 2024, 16(1).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTirumuru N, Zhao BS, Lu W, Lu Z, He C, Wu L. N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression. eLife 2016, 5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLichinchi G, Gao S, Saletore Y, Gonzalez GM, Bansal V, Wang Y, Mason CE, Rana TM. Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells. Nat Microbiol. 2016;1:16011.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu W, Tirumuru N, St Gelais C, Koneru PC, Liu C, Kvaratskhelia M, He C, Wu L. N(6)-Methyladenosine-binding proteins suppress HIV-1 infectivity and viral production. J Biol Chem. 2018;293(34):12992\u0026ndash;3005.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen S, Kumar S, Espada CE, Tirumuru N, Cahill MP, Hu L, He C, Wu L. N6-methyladenosine modification of HIV-1 RNA suppresses type-I interferon induction in differentiated monocytic cells and primary macrophages. PLoS Pathog. 2021;17(3):e1009421.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMishra T, Phillips S, Zhao Y, Wilms B, He C, Wu L. Epitranscriptomic m(6)A modifications during reactivation of HIV-1 latency in CD4(+) T cells. mBio. 2024;15(11):e0221424.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMishra T, Phillips S, Maldonado C, Stapleton JT, Wu L. Antiretroviral Therapy Suppresses RNA N(6)-Methyladenosine Modification in Peripheral Blood Mononuclear Cells from HIV-1-Infected Individuals. AIDS Res Hum Retroviruses. 2024;40(9):511\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShiels MS, Pfeiffer RM, Gail MH, Hall HI, Li J, Chaturvedi AK, Bhatia K, Uldrick TS, Yarchoan R, Goedert JJ, et al. Cancer burden in the HIV-infected population in the United States. J Natl Cancer Inst. 2011;103(9):753\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChao C, Leyden WA, Xu L, Horberg MA, Klein D, Towner WJ, Quesenberry CP Jr., Abrams DI, Silverberg MJ. Exposure to antiretroviral therapy and risk of cancer in HIV-infected persons. AIDS. 2012;26(17):2223\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHernandez-Ramirez RU, Shiels MS, Dubrow R, Engels EA. Cancer risk in HIV-infected people in the USA from 1996 to 2012: a population-based, registry-linkage study. Lancet HIV. 2017;4(11):e495\u0026ndash;504.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin S, Choe J, Du P, Triboulet R, Gregory RI. The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells. Mol Cell. 2016;62(3):335\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeng H, Huang H, Wu H, Qin X, Zhao BS, Dong L, Shi H, Skibbe J, Shen C, Hu C, et al. METTL14 Inhibits Hematopoietic Stem/Progenitor Differentiation and Promotes Leukemogenesis via mRNA m(6)A Modification. Cell Stem Cell. 2018;22(2):191\u0026ndash;205. e199.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eParis J, Morgan M, Campos J, Spencer GJ, Shmakova A, Ivanova I, Mapperley C, Lawson H, Wotherspoon DA, Sepulveda C, et al. Targeting the RNA m(6)A Reader YTHDF2 Selectively Compromises Cancer Stem Cells in Acute Myeloid Leukemia. Cell Stem Cell. 2019;25(1):137\u0026ndash;e148136.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWinkler R, Gillis E, Lasman L, Safra M, Geula S, Soyris C, Nachshon A, Tai-Schmiedel J, Friedman N, Le-Trilling VTK, et al. Publisher Correction: m6A modification controls the innate immune response to infection by targeting type I interferons. Nat Immunol. 2019;20(2):243.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMishra T, Phillips S, Wu L. Method for the Enrichment of N(6)-Methyladenosine-Modified Cellular and HIV-1 RNA. Methods Mol Biol. 2024;2807:195\u0026ndash;208.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAsgari MM, Ray GT, Quesenberry CP Jr., Katz KA, Silverberg MJ. Association of Multiple Primary Skin Cancers With Human Immunodeficiency Virus Infection, CD4 Count, and Viral Load. JAMA Dermatol. 2017;153(9):892\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNicolau IA, Moineddin R, Brooks JD, Antoniou T, Gillis JL, Kendall CE, Cooper C, Cotterchio M, Salters K, Smieja M, et al. Associations of CD4 Cell Count Measures With Infection-Related and Infection-Unrelated Cancer Risk Among People With HIV. J Acquir Immune Defic Syndr. 2024;96(5):447\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai S, Zhou J, Luo X, Zhang C, Jin S, Ren J, Cui J. Phase transition of WTAP regulates m6A modification of interferon-stimulated genes. \u003cem\u003eeLife\u003c/em\u003e 2025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan X, Zhu Y, Ke J, Zhai Y, Huang M, Zhang X, He H, Zhang X, Zhao X, Guo K, et al. Progression of m(6)A in the tumor microenvironment: hypoxia, immune and metabolic reprogramming. Cell Death Discov. 2024;10(1):331.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eComoglio PM, Trusolino L, Boccaccio C. Known and novel roles of the MET oncogene in cancer: a coherent approach to targeted therapy. Nat Rev Cancer. 2018;18(6):341\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDai L, Trillo-Tinoco J, Cao Y, Bonstaff K, Doyle L, Del Valle L, Whitby D, Parsons C, Reiss K, Zabaleta J, et al. Targeting HGF/c-MET induces cell cycle arrest, DNA damage, and apoptosis for primary effusion lymphoma. Blood. 2015;126(26):2821\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe WQ, Pang W, Li N, Li AQ, Li YH, Lu Y, Shen F, Xin R, Song TZ, Tian RR, et al. IFI27 inhibits HIV-1 replication by degrading Gag protein through the ubiquitin-proteasome pathway. J Virol. 2024;98(11):e0135624.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang S, Zhao J, Song J, Li Y, Zuo R, Sa Y, Ma Z, OuYang H. Interferon alpha-inducible protein 27 (IFI27) is a prognostic marker for pancreatic cancer based on comprehensive bioinformatics analysis. Bioengineered. 2021;12(1):8515\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchoggins JW, Wilson SJ, Panis M, Murphy MY, Jones CT, Bieniasz P, Rice CM. A diverse range of gene products are effectors of the type I interferon antiviral response. Nature. 2011;472(7344):481\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZicari S, Sessa L, Cotugno N, Ruggiero A, Morrocchi E, Concato C, Rocca S, Zangari P, Manno EC, Palma P. Immune Activation, Inflammation, and Non-AIDS Co-Morbidities in HIV-Infected Patients under Long-Term ART. \u003cem\u003eViruses\u003c/em\u003e 2019, 11(3).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang C, Chen J, Ren J, Li X, Zhang Y, Huang B, Xu Y, Dong L, Cao Y. N(6)-methyladenosine levels in peripheral blood RNA: a potential diagnostic biomarker for colorectal cancer. Cancer Cell Int. 2024;24(1):96.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTirumuru N, Wu L. HIV-1 envelope proteins up-regulate N(6)-methyladenosine levels of cellular RNA independently of viral replication. J Biol Chem. 2019;294(9):3249\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRinaldi S, Pallikkuth S, Cameron M, de Armas LR, Cotugno N, Dinh V, Pahwa R, Richardson B, Saini SR, Rocca S, et al. Impact of Early Antiretroviral Therapy Initiation on HIV-Specific CD4 and CD8 T Cell Function in Perinatally Infected Children. J Immunol. 2020;204(3):540\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFranceschi S, Lise M, Clifford GM, Rickenbach M, Levi F, Maspoli M, Bouchardy C, Dehler S, Jundt G, Ess S, et al. Changing patterns of cancer incidence in the early- and late-HAART periods: the Swiss HIV Cohort Study. Br J Cancer. 2010;103(3):416\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePing XL, Sun BF, Wang L, Xiao W, Yang X, Wang WJ, Adhikari S, Shi Y, Lv Y, Chen YS, et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 2014;24(2):177\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDu Y, Hou G, Zhang H, Dou J, He J, Guo Y, Li L, Chen R, Wang Y, Deng R, et al. SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function. Nucleic Acids Res. 2018;46(10):5195\u0026ndash;208.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu F, Wei J, Cui X, Yu C, Ni W, Bungert J, Wu L, He C, Qian Z. Post-translational modification of RNA m6A demethylase ALKBH5 regulates ROS-induced DNA damage response. Nucleic Acids Res. 2021;49(10):5779\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDai XY, Shi L, Li Z, Yang HY, Wei JF, Ding Q. Main N6-Methyladenosine Readers: YTH Family Proteins in Cancers. Front Oncol. 2021;11:635329.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMackelprang RD, Filali-Mouhim A, Richardson B, Lefebvre F, Katabira E, Ronald A, Gray G, Cohen KW, Klatt NR, Pecor T, et al. Upregulation of IFN-stimulated genes persists beyond the transitory broad immunologic changes of acute HIV-1 infection. iScience. 2023;26(4):106454.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJi M, Ganesan S, Xia B, Huo Y. Targeting c-MET Alterations in Cancer: A Review of Genetic Drivers and Therapeutic Implications. Cancers (Basel) 2025, 17(9).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAtaie-Kachoie P, Pourgholami MH, Morris DL. Inhibition of the IL-6 signaling pathway: a strategy to combat chronic inflammatory diseases and cancer. Cytokine Growth Factor Rev. 2013;24(2):163\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeissl K, Macho-Maschler S, Muller M, Strobl B. The good and the bad faces of STAT1 in solid tumours. Cytokine. 2017;89:12\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen RH, Du Y, Han P, Wang HB, Liang FY, Feng GK, Zhou AJ, Cai MY, Zhong Q, Zeng MS, et al. ISG15 predicts poor prognosis and promotes cancer stem cell phenotype in nasopharyngeal carcinoma. Oncotarget. 2016;7(13):16910\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang H, Lv J, Huang Y, Mo Z, Xu H, Huang Y, Yang L, Wu Z, Li H, Qin Y. IFI27 is a potential therapeutic target for HIV infection. Ann Med. 2022;54(1):314\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcFadden MJ, McIntyre ABR, Mourelatos H, Abell NS, Gokhale NS, Ipas H, Xhemalce B, Mason CE, Horner SM. Post-transcriptional regulation of antiviral gene expression by N6-methyladenosine. Cell Rep. 2021;34(9):108798.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong H, Song J, Cheng M, Zheng M, Wang T, Tian S, Flavell RA, Zhu S, Li HB, Ding C, et al. METTL3-mediated m(6)A RNA methylation promotes the anti-tumour immunity of natural killer cells. Nat Commun. 2021;12(1):5522.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuirguis AA, Ofir-Rosenfeld Y, Knezevic K, Blackaby W, Hardick D, Chan YC, Motazedian A, Gillespie A, Vassiliadis D, Lam EYN, et al. Inhibition of METTL3 Results in a Cell-Intrinsic Interferon Response That Enhances Antitumor Immunity. Cancer Discov. 2023;13(10):2228\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarmona-Perez L, Dagenais-Lussier X, Mai LT, Stogerer T, Swaminathan S, Isnard S, Rice MR, Barnes BJ, Routy JP, van Grevenynghe J et al. The TLR7/IRF-5 axis sensitizes memory CD4\u0026thinsp;+\u0026thinsp;T cells to Fas-mediated apoptosis during HIV-1 infection. JCI Insight 2023, 8(13).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUrban-Wojciuk Z, Khan MM, Oyler BL, Fahraeus R, Marek-Trzonkowska N, Nita-Lazar A, Hupp TR, Goodlett DR. The Role of TLRs in Anti-cancer Immunity and Tumor Rejection. Front Immunol. 2019;10:2388.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HIV-1 infection, cancer, people living with HIV-1 (PLWH), N6-methyladenosine (m6A), peripheral blood mononuclear cells, type-I interferon (IFN-I), IFN-responsive genes, antiretroviral therapy","lastPublishedDoi":"10.21203/rs.3.rs-8070380/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8070380/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"(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.","manuscriptTitle":"Elevated m 6 A RNA Modifications Associate with Immune Dysregulation and Cancer in People with HIV-1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 13:34:14","doi":"10.21203/rs.3.rs-8070380/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-05T12:29:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-03T22:35:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282899791218349736006244596844037261279","date":"2026-01-25T23:49:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-29T17:41:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104001093083117622899925264218124121601","date":"2025-11-17T03:01:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33171180234563651161746119916047075752","date":"2025-11-16T16:38:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-15T00:01:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-14T23:54:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-13T22:02:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-12T22:48:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-11-12T22:44:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"704f1e4f-be5c-4c1b-8bc9-4b8951e29e2a","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:10:39+00:00","versionOfRecord":{"articleIdentity":"rs-8070380","link":"https://doi.org/10.1186/s12885-026-15874-x","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2026-03-21 15:57:41","publishedOnDateReadable":"March 21st, 2026"},"versionCreatedAt":"2026-01-13 13:34:14","video":"","vorDoi":"10.1186/s12885-026-15874-x","vorDoiUrl":"https://doi.org/10.1186/s12885-026-15874-x","workflowStages":[]},"version":"v1","identity":"rs-8070380","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8070380","identity":"rs-8070380","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}