Title

Assessment of monovalent and bivalent SMAC mimetics to both shock and kill the HIV reservoir

Presenter

Kiho Tanaka

Authors

K. Tanaka¹, Y. Kim¹, C. Tumpach¹, J. Ong¹, M. Roche¹, M. Pelligrini², S. Lewin^1,3,4

Institutions

¹The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Department of Infectious Diseases, Melbourne, Australia, ²The Walter and Eliza Hall Institute of Medical Research, Infectious Diseases and Immune Defence, Melbourne, Australia, ³Royal Melbourne Hospital at The Peter Doherty Institute for Infection and Immunity, Victorian Infectious Diseases Service, Melbourne, Australia, ⁴Alfred Hospital and Monash University, Department of Infectious Diseases, Melbourne, Australia

BACKGROUND: Latently infected CD4+ T-cells are primed for survival due to the over-expression of factors that regulate apoptosis such as inhibitors of apoptosis proteins (IAPs). This may explain why ‘shock and kill’ efforts to date have largely failed. SMAC mimetics (SMACm) have been shown by others to increase binding of non-canonical NfkB (ncNFkB) complex to the HIV long terminal repeat and can potently reverse HIV latency. SMACm can also inhibit IAPs leading to an increase in tumor necrosis factor alpha (TNF-a) mediated extrinsic apoptosis. We hypothesised that SMACm would increase death of latently infected cells in the presence of TNF-a.
METHODS: We assessed bivalent (AZD5582, BV6 and Birinapant) and monovalent (GDC0152, GDC0197, xevinapant and LCL161) SMACm in the presence and absence of TNF-a (20ng/ml) in peripheral blood mononuclear cells (PBMC) and sorted CD4+ T-cells from uninfected donors and people with HIV (PWH) on antiretroviral therapy. Latency reversal was assessed using a cell line containing integrated HIV with a mutation in env and vpr and a green fluorescent protein (GFP) reporter (J-Lat clone 10.6). GFP expression and cell death using a live dead stain were quantified by flow cytometry. NFkB activation and cIAP1 degradation was measured by western blot.
RESULTS: The bivalent compared to monovalent SMACm showed higher levels of GFP expression in J-Lat10.6 (100nM; mean±SEM GFP expression 1.3%±0.14% vs 6.0%±1.9% respectively, p=0.03) and induced greater cIAP1 degradation and induction of p52 from p100 cleavage in PBMC from uninfected donors. Both bivalent and monovalent SMACm demonstrated similar toxicity profiles in CD4+ T-cells isolated from uninfected PBMC (100nM; 8.5%±3.3% vs 9.9%±0.4% cell death, p=0.52). Addition of TNF-a to SMACm led to no difference in cell toxicity and cIAP1 degradation in CD4+T-cells from uninfected donors and PWH.
CONCLUSIONS: Bivalent compared to monovalent SMACm induce greater latency reversal and also cell death in cell lines but have similar levels of toxicity in primary cells. The additional of TNF-a did not increase cell death. Further work is continuing on the impact of these compounds on CD4+ T-cells from PWH on ART.

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