Dissertation/Thesis Abstract

Data-Driven Analysis of Phospho-Signaling Network Responses Enables Latent HIV Infected T Cell Targeting
by Fong, Linda Ellen, Ph.D., Yale University, 2018, 213; 10957324
Abstract (Summary)

Viral latency remains the most significant obstacle to HIV eradication. Current clinical strategies aim to purge the latent CD4+ T cell reservoir by activating viral expression, but are undercut by the inability to clear the latent reservoir. We first evaluated co-drugging criteria in a quantitative manner to optimize viral expression. However, this approach faces many challenges; and thus, we proposed to identify and target dysregulated signaling pathways in latent HIV-infected cells to promote cell death as a novel approach for eradication. To identify how HIV latency and reactivation alter signal transduction pathways regulating cell death, we explored the acute signaling response of latent HIV-infected CD4+ T cells across in vitro human latency models using systems-level analyses. We measured phosphorylation of five signaling proteins (AKT, IKBa, ERK, p38, and JNK) after stimulation with T-cell activating agents or latency reversing agents in infected cells and uninfected cells. Using these phosphorylation signatures, we built data-driven statistical models that successfully classified infected and uninfected cells, demonstrating that latent infection alters signaling at a systems level. We further identified that the stress kinase pathways p38 and JNK exhibited elevated signaling in latently infected cells and could be targeted to specifically increase cell death, independent of HIV reactivation.

To work out the mechanisms by which latent and reactivating HIV alters cell death regulation, we further examined signaling of 31 proteins in single cells over 48 hours using mass cytometry. Mass cytometry provides measurements at single-cell resolution, enabling us to separate responses in cells with latent versus reactivating HIV based on viral expression. We used conditional density-based analysis of the single-cell data to quantify the strength of signaling activity along different pathways. We discovered that latent and HIV-expressing cells are sensitized to apoptotic cell death via activation of p38-p53 signaling and inhibition of AKT/mTOR signaling. We identified a novel interaction in infected cells, in which increased p38 signaling activates the pro-death activity of the protein BAD, leading to increased apoptosis. Finally, we show in vitro that p38 and AKT/mTOR pathways can be simultaneously targeted to deplete the latent reservoir by preferentially killing latently infected cells without viral activation. Overall, we demonstrate that targeting altered phosphorylation signatures of latent HIV-infected cells provides a novel and effective strategy for latent HIV eradication.

Indexing (document details)
Advisor: Miller-Jensen, Kathryn
Commitee:
School: Yale University
School Location: United States -- Connecticut
Source: DAI-B 79/12(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Caribbean Studies, Biomedical engineering, Immunology
Keywords: Cell Signaling, HIV Latency, High-Dimentional Analysis, Mass Cytometry, Network Dysregulation, Systems Biology
Publication Number: 10957324
ISBN: 9780438273696
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