DNA-PK-mediated regulation of inflammation controls aging

Chronic inflammation is a critical driver and hallmark of the aging process, and its severity is exacerbated in people with HIV (PWH), despite effective antiretroviral therapy (ART). We discovered the essential role of DNA-dependent protein kinase (DNA-PK) in HIV life cycle. We found that HIV infection exposure activates DNA-PK (p-DNA-PK, S2056) and TRIM28 (p-TRIM28, S824). Recent evidence suggests that TRIM28 stimulation enhances NOD-like receptor protein 3 (NLRP3) inflammasome activation by preventing the ubiquitination and proteasomal degradation of NLRP3 protein through TRIM28-mediated SUMOylation of NLRP3. Therefore, targeting DNA-PK may offer a dual mechanism to restrict both viral load and the induction of inflammation associated with HIV, in addition to HIV persistence. The objective of this study is to delineate the molecular mechanisms by which HIV promotes aging by intensifying neuroinflammation via DNA-PK-induced TRIM28-mediated regulation of NLRP3 inflammasome activation and to test the therapeutic potential of two in-human tested DNA-PK inhibitors (DNA-PKi) in limiting neuroinflammation in a suitable humanized mouse model. Thus, the study will address the gap in knowledge regarding our understanding of the factors that drive neuroinflammation and premature aging in the setting of HIV and effective ART, the main target area of the NOFO, and have both basic and clinical implications. Based on our numerous publications on this subject, we have hypothesized that HIV-stimulated DNA-PK triggers neuroinflammation and the aging process, both by enhancing HIV gene expression and triggering the activation of NLRP3 inflammasome, through TRIM28 activation. Clinically tested DNA-PKi can mitigate neuroinflammation and aging by inducing NLRP3 degradation. In Aim 1, we will define the mechanism by which DNA-PK-induced TRIM28 restricts NLRP3 ubiquitination and degradation in the context of HIV +/- ART in iPSC-derived human microglial cells (IDMs). In Aim 2, we will evaluate the impact of inhibiting the DNA-PK-TRIM28-NLRP3 axis in restricting inflammasome activation and the aging process (DNA methylation clock) in the setting of HIV and ART. In Aim 3, we will test the therapeutic potential of two in-human tested DNA-PKi in limiting inflammation, HIV infection, reservoir, and rebound in a humanized mouse model +/- HIV +/- ART. Upon conclusion of the study, we will define the molecular mechanisms by which HIV exacerbates neuroinflammation and test the therapeutic potential of clinically tested DNA-PKi in preventing HIV-mediated inflammation of PWH, even on effective ART. The proposed research is innovative because data will be obtained using different systemic approaches, including at a single-cell level, and the use of an appropriate mouse model. This contribution is significant, as we will test the clinical relevance of DNA-PKi for designing optimized therapies targeting both HIV persistence and associated comorbidities, such as aging, in PWH under effective ART.