PROJECT SUMMARY When people encounter an infectious agent or a vaccine for the first time, they develop an immune response to that agent or vaccine. Some of the respondent cells may persist for years ? even decades, becoming memory T cells, so that if the infectious agent is again encountered, the subsequent immune response can be rapid and exact. Since these cells must to persist for long periods, they have developed mechanisms to resist the normal process of cell turnover, i.e., these cells withstand death signals. HIV infection is a chronic, manageable disease that, as yet, cannot be cured due to the persistence of HIV in memory CD4 T cells that are resistant to cell turnover and death. In T cells that are not memory CD4 T cells, replicating HIV produces HIV proteins, which are toxic and kill the T cell. Paradoxically, when HIV is reactivated in memory CD4 T cells, these same HIV proteins do not kill the cell. We believe the reason these memory CD4 T cells do not die after HIV is reactivated, is that they are intrinsically resistant to cell death. Accordingly there are multiple levels of regulation within cells that together determine whether a cell will, or will not undergo cell death. In broad terms in order for a cell to undergo apoptosis (one form of cell death) two conditions must be met: (i) a pro-apoptotic stimulus, and (ii) a permissive cellular environment, where anti-apoptotic machinery does not prevent propagation of the pro-apoptotic signal. Our laboratory group has studied how HIV causes the death of CD4 T cells, focusing on the HIV protease- Casp8p41 pathway. During the course of this grant's last funding cycle, we discovered that Casp8p41 kills cells by binding to the pro-apoptotic protein Bak. Conversely, if Casp8p41 binds the anti-apoptotic protein Bcl2, then HIV does not kill a cell, instead becoming a persistent, latently-infected reservoir cell. We have generated novel and clinically relevant observations that antagonizing Bcl2 with a Bcl2 inhibitor (venetoclax) alters the number of infected cells that die following HIV reactivation, reducing the HIV reservoir size. Similarly, we have shown that after Casp8p41 is bound by Bcl2, the complex is polyubiquitinated and degraded by the proteasome. Consequently, using clinically available proteasome inhibitors (bortezomib or ixazomib) causes accumulation of Casp8p41, enhances infected cell killing, alters the number of dead infected cells following HIV reactivation, and effectively reduces the HIV reservoir size. The focus of the current application is to optimize these treatment approaches to: (i) test combinations of Bcl2 antagonists with optimized latency- reversal strategies, (ii) test the effect of the most promising combinations on the lymphoid HIV reservoir, and (iii) determine why some, but not all, patients cells respond to Bcl2 inhibition, testing the hypothesis that polymorphisms, or differences in expression of key proteins involved in this pathway, differ between responders and nonresponders.
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