Ex vivo HIV DNA integration in STAT3 drives T cell persistence—A model of HIV-associated T cell lymphoma

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Abstract
Oncogenic retroviruses are known for their pathogenesis via insertional mutagenesis, in which the presence of a provirus and its transcriptional control elements alter the expression of a nearby or surrounding host gene. There are reports of proviral integration driving oncogenesis in people with HIV and the use of HIV-derived vectors for gene therapy has raised concern about oncogenic side effects. To study this issue, we used an ex vivo human CD4+ T cell infection model developed in our laboratory to identify HIV-1 integration sites that might influence cell proliferation or survival. Combining integration site analysis and bulk RNA sequencing, we established that an upregulated STAT3 signature due to proviral insertional mutagenesis was associated with persistent HIV-infected CD4+ T cells. HIV+ persistent cells also expressed a STAT3-related anti-apoptotic and cytotoxic phenotype that resembles that of HIV-associated T cell lymphomas. HIV insertional mutagenesis of STAT3 and expression of its downstream targets provides a model of HIV-associated T cell lymphomas that can be used to further determine the oncogenic drivers of HIV-associated lymphomas, both AIDS- and gene therapy-associated, and, potentially, to evaluate therapeutics against these HIV-associated cancers. Author Summary: The effects of HIV proviral insertional mutagenesis have been demonstrated in a handful of HIV-associated T cell lymphomas, where integration of an HIV provirus within intron 1 of STAT3 , results in increased expression of the STAT3 protein. To study the effects of HIV insertional mutagenesis, we established an ex vivo culture protocol of primary human CD4+ T cells infected with a replication-incompetent HIV vector with a gfp-reporter. After infection, the HIV/GFP+ cells from all three donors declined, but, over time, 3/6 replicates from one donor populations of infected cells rebounded. The resurgent HIV/GFP+ cells contained a provirus integrated within intron 1 of STAT3 , which led to increases in gene expression, STAT3 activation, and upregulation of a STAT3 -associated anti-apoptotic and cytotoxic phenotype. The STAT3 -associated gene signature shared similarities to the HIV-associated lymphomas with similar integration sites. Additionally, in all 3 replicates, insertional mutagenesis of genes other than STAT3 may have also contributed to clonal expansion of HIV/GFP+ T cells. Overall, we have demonstrated that HIV provirus insertional mutagenesis can influence T cell persistence. Our study provides a primary T cell culture model system that can be used to further study how proviral insertional mutagenesis influences HIV-associated T cell lymphomas and the safety of lentiviral vectors used in gene and cell therapies.
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