BRING-SynBio: Deciphering and directing lactylation epigenetics to control macrophage immune cell phenotype

University Of Delaware, Newark DE

Investigators

Abstract

Macrophages are immune cells that play critical roles in fighting disease, healing tissues, and maintaining overall health. Their behavior can shift in ways that either help or harm the body, depending on their environment. Histone lactylation, in which lactate, a small molecule metabolite produced in the body, changes how genes are turned on or off, may help explain how these cells change roles. This project will use synthetic biology to create precision tools that can add or remove lactylation marks on DNA-packaging proteins. These tools will help scientists understand how lactylation affects macrophage behavior and could lead to new ways to control immune responses in diseases like cancer. Course module development and support of undergraduate researchers will help to grow the biomanufacturing workforce. This project investigates the functional role of histone lactylation in macrophage polarization. Lactylation has been linked to macrophage transitions from inflammatory (M1) to anti-inflammatory (M2) phenotypes, particularly within the immunosuppressive tumor microenvironment. The central hypothesis is that modulating histone lactylation can selectively control macrophage phenotype, independent of external stimuli. In Phase I, lactylation writers and erasers will be created. These are enzymes capable of adding or removing lactylation at specific sites on histones. First, a detailed map of histone lactylation across macrophage polarization states and external stimuli will be established. Site-specific lactylation tools will be generated by engineering a dCas9-p300 fusion system to promote lactylation at H3K18 near the Arg1 gene and evolved using a yeast-based screening platform for increased lactylation selectivity. Histone deacetylase complexes using CRISPR-Cas9 strategies to remove lactylation will be designed and implemented, and the impact on macrophage phenotype assessed. Key milestones for Phase 1 include validation of lactylation writers and erasers with confirmed selectivity over acetylation and demonstration that these tools can meaningfully regulate macrophage phenotype. In Phase 2, the functional impact of these new lactylation tools in preclinical cancer models will be evaluated. Studies will employ lactylation readers and writers to quantitate how lactylation influences tumor-associated macrophage repolarization and how these tools can improve outcomes of chimeric antigen receptor macrophages, using both in vitro and in vivo tumor environments. In total, this project will advance fundamental understanding of macrophage immunometabolism, provide versatile tools for lactylation engineering, and lay the groundwork for durable macrophage-based therapies for cancer and other immune-mediated diseases. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.