title: Identification of proximal biomarkers of PKC agonism and evaluation of their role in HIV reactivation.
abstract: METHODS
  The HIV latent CD4+T cell reservoir is broadly recognized as a barrier to HIV cure. Induction of HIV expression using protein kinase C (PKC) agonists is one approach under investigation for reactivation of latently infected CD4+T cells (Beans et al., 2013; Abreu et al., 2014; Jiang et al., 2014; Jiang and Dandekar, 2015). We proposed that an increased understanding of the molecular mechanisms of action of PKC agonists was necessary to inform on biological signaling and pharmacodynamic biomarkers. RNA sequencing (RNA Seq) was applied to identify genes and pathways modulated by PKC agonists.
  
  
  METHODS
  Human CD4+T cells were treated ex vivo with Phorbol 12-myristate 13-acetate, prostatin or ingenol-3-angelate. At 3 h and 24 h post-treatment, cells were harvested and RNA-Seq was performed on RNA isolated from cell lysates. The genes differentially expressed across the PKC agonists were validated by quantitative RT-PCR (qPCR). A subset of genes was evaluated for their role in HIV reactivation using siRNA and CRISPR approaches in the Jurkat latency cell model.
  
  
  RESULTS
  Treatment of primary human CD4+T cells with PKC agonists resulted in alterations in gene expression. qPCR of RNA Seq data confirmed upregulation of 24 genes, including CD69, Egr1, Egr2, Egr3, CSF2, DUSP5, and NR4A1. Gene knockdown of Egr1 and Egr3 resulted in reduced expression and decreased HIV reactivation in response to PKC agonist treatment, indicating a potential role for Egr family members in latency reversal.
  
  
  CONCLUSIONS
  Overall, our results offer new insights into the mechanism of action of PKC agonists, biomarkers of pathway engagement, and the potential role of EGR family in HIV reactivation.
authors.affiliation: Infectious Diseases and Vaccines, Merck & Co., West Point, PA, USA.
authors.forename: Sai Vikram
authors.initials: SV
authors.lastname: Vemula
authors.affiliation: Infectious Diseases and Vaccines, Merck & Co., West Point, PA, USA.
authors.forename: Jill W
authors.initials: JW
authors.lastname: Maxwell
authors.affiliation: Discovery Pharmacogenomics, Merck & Co., West Point, PA, USA.
authors.forename: Alexey
authors.initials: A
authors.lastname: Nefedov
authors.affiliation: Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., West Point, PA, USA.
authors.forename: Bang-Lin
authors.initials: BL
authors.lastname: Wan
authors.affiliation: Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., West Point, PA, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Steve
authors.affiliation: Infectious Diseases and Vaccines, Merck & Co., West Point, PA, USA.
authors.forename: William
authors.initials: W
authors.lastname: Newhard
authors.affiliation: Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., West Point, PA, USA.
authors.forename: Rosa I
authors.initials: RI
authors.lastname: Sanchez
authors.affiliation: Medicinal Chemistry, Merck & Co., West Point, PA, USA.
authors.forename: David
authors.initials: D
authors.lastname: Tellers
authors.affiliation: Infectious Diseases and Vaccines, Merck & Co., West Point, PA, USA.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Barnard
authors.affiliation: Infectious Diseases and Vaccines, Merck & Co., West Point, PA, USA.
authors.forename: Wade
authors.initials: W
authors.lastname: Blair
authors.affiliation: Infectious Diseases and Vaccines, Merck & Co., West Point, PA, USA.
authors.forename: Daria
authors.initials: D
authors.lastname: Hazuda
authors.affiliation: Discovery Pharmacogenomics, Merck & Co., West Point, PA, USA.
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Webber
authors.affiliation: Infectious Diseases and Vaccines, Merck & Co., West Point, PA, USA. Electronic address: bonnie_howell@merck.com.
authors.forename: Bonnie J
authors.initials: BJ
authors.lastname: Howell
mesh_terms: D015415:Biomarkers; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000821:virology; D002478:Cells, Cultured; D004224:Diterpenes / Q000737:chemistry / Q000494:pharmacology; D054313:Drug Agonism; D051766:Early Growth Response Protein 1 / Q000235:genetics; D051776:Early Growth Response Protein 3 / Q000235:genetics; D015870:Gene Expression; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D019169:Jurkat Cells; D008297:Male; D010704:Phorbols / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D014775:Virus Activation / Q000187:drug effects*; D017735:Virus Latency / Q000187:drug effects*
chemical_list: D015415:Biomarkers; D004224:Diterpenes; C497325:EGR1 protein, human; C079945:EGR3 protein, human; D051766:Early Growth Response Protein 1; D010704:Phorbols; D051776:Early Growth Response Protein 3; D011493:Protein Kinase C; C008125:ingenol; C033085:phorbol
keywords: Biomarkers; HIV latency; Protein kinase C agonists