An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation.

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Acetyl-Click Screening Platform Identifies Small-Molecule Inhibitors of Histone Acetyltransferase 1 (HAT1).

HAT1 is a central regulator of chromatin synthesis that acetylates nascent histone H4. To ascertain whether targeting HAT1 is a viable anticancer treatment strategy, we sought to identify small-molecule inhibitors of HAT1 by developing a high-throughput HAT1 acetyl-click assay. Screening of small-molecule libraries led to the discovery of multiple riboflavin analogs that inhibited HAT1 enzymatic activity. Compounds were refined by synthesis and testing of over 70 analogs, which yielded structure-activity relationships. The isoalloxazine core was required for enzymatic inhibition, whereas modifications of the ribityl side chain improved enzymatic potency and cellular growth suppression. One compound (JG-2016 [24a]) showed relative specificity toward HAT1 compared to other acetyltransferases, suppressed the growth of human cancer cell lines, impaired enzymatic activity in cellulo, and interfered with tumor growth. This is the first report of a small-molecule inhibitor of the HAT1 enzyme complex and represents a step toward targeting this pathway for cancer therapy.

Polycomb deficiency drives a FOXP2-high aggressive state targetable by epigenetic inhibitors.

Inhibitors of the Polycomb Repressive Complex 2 (PRC2) histone methyltransferase EZH2 are approved for certain cancers, but realizing their wider utility relies upon understanding PRC2 biology in each cancer system. Using a genetic model to delete Ezh2 in KRAS-driven lung adenocarcinomas, we observed that Ezh2 haplo-insufficient tumors were less lethal and lower grade than Ezh2 fully-insufficient tumors, which were poorly differentiated and metastatic. Using three-dimensional cultures and in vivo experiments, we determined that EZH2-deficient tumors were vulnerable to H3K27 demethylase or BET inhibitors. PRC2 loss/inhibition led to de-repression of FOXP2, a transcription factor that promotes migration and stemness, and FOXP2 could be suppressed by BET inhibition. Poorly differentiated human lung cancers were enriched for an H3K27me3-low state, representing a subtype that may benefit from BET inhibition as a single therapy or combined with additional EZH2 inhibition. These data highlight diverse roles of PRC2 in KRAS-driven lung adenocarcinomas, and demonstrate the utility of three-dimensional cultures for exploring epigenetic drug sensitivities for cancer.

Pandora's Box: Disseminated Coccidioidomycosis Associated with Self-Medication with an Unregulated Potent Corticosteroid Acquired in Mexico.

Coccidioidomycosis (CM), caused by the dimorphic fungi Coccidioides immitis and C. posadasii, typically presents as acute or chronic pulmonary disease. However, disseminated disease occurs in about 1% of patients. Disseminated CM may affect multiple organ systems, including cutaneous, osteoarticular, and central nervous system sites. Here, we present a case of disseminated CM in a patient from a border city in Texas. The patient had a history of uncontrolled diabetes mellitus and was also taking an over-the-counter medication acquired in Mexico that contained a potent corticosteroid. The patient presented with seizures and was found to have a brain infarct, cavitary lung lesions, synovitis of the knee, multiple skin lesions, and chorioretinitis. The patient had a very high complement fixation titer for Coccidioides; fungal spherules were seen in a skin biopsy specimen, and Coccidioides grew in culture from a sample of synovial fluid and the skin biopsy specimen. This case illustrates the dissemination potential of Coccidioides, the danger of unregulated pharmaceuticals, the importance of thorough history taking, and recognizing risk factors that contribute to disseminated CM.