IMPACT OF BCL2 PHOSPHORYLATION ON HIV DYNAMICS, RESERVOIR SIZE AND SUSCEPTIBILITY TO VENETOCLAX

PROJECT SUMMARY
Following HIV infection, a subset of T cells do not die and can revert to a latent state of infection. Much
research over the past decade has consistently found that cells which persist are enriched for the antiapoptotic
protein Bcl2. Consequently, many groups have been studying the selective Bcl2 antagonist Venetoclax and
have now shown that Venetoclax augments death of HIV infected cells during acute HIV infection, IL-7 induced
homeostatic proliferation, during reactivation from latency alone and in the presence of autologous HIV specific
T cells, and in a humanized mouse model of acute HIV infection where Venetoclax monotherapy caused
greater killing of HIV infected cells, normalized CD4:CD8 ratios, and reduced viral load and HIV reservoir size.
Like many proteins, Bcl2 can undergo post translational modification by phosphorylation which alters the ability
of Bcl2 to bind death inducing proteins such as Casp8p41, which is an HIV specific death stimulus. Bcl2
phosphorylation also alters the ability of Venetoclax to bind Bcl2 and promote HIV clearance. In this
application, we present preliminary and novel data showing that during HIV infection, both in vitro and in
lymphoid tissues from HIV infected subjects, Bcl2 is pervasively phosphorylated.
To comprehensively study the effects of Bcl2 phosphorylation of HIV replication kinetics, cell killing and
Venetoclax effects, we have generated for the first time a Bcl2 CRISPR knockout Jurkat cell line and have also
generated Jurkats which stably express different Bcl2 variants with phospho mimic substitutions, or phospho
resistant substitutions at residues known to be susceptible to phosphorylation (at positions 56, 69, 70, 74, 87).
Initial results using those cells indicates that phospho Bcl2 cells are more resistant to HIV induced cell death
and are resistant to the anti-HIV effects of Venetoclax.
Given that Venetoclax has anti-HIV and anti-reservoir effects, it is highly relevant to understand the
determinants of Venetoclax effectiveness and, more broadly, determinants of Bcl2 function. Studies proposed
in this application will define the mechanism and sites of Bcl2 phosphorylation, the impact of those
phosphorylation events of HIV replication kinetics, the number of HIV DNA positive cells, and the anti-HIV
effects of Venetoclax. In addition, we will test clinically relevant strategies to overcome the functional
consequences of phosphorylated Bcl2, enabling more HIV infected cells to die after reactivation from latency.

Grant Number: 1R56AI183801-01
NIH Institute/Center: NIH
Principal Investigator: ANDREW BADLEY