Antibody-drug conjugates (ADCs) selectively deliver highly toxic chemotherapeutic agents to target antigen-expressing cells and have become an important cancer treatment in recent years.However, the molecular mechanisms by which ADCs are internalized and activated within cells remain unclear. Here we use CRISPR-Cas9 screens to identify genes that control the toxicity of ADCs. Our results demonstrate critical roles for a range of known and novel endolysosomal trafficking regulators in ADC toxicity. We identify and characterize C18orf8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of CRISPR screens with ADCs bearing a noncleavable linker versus a cleavable valine-citrulline (VC) linker, we show that a subset of late endosomal and lysosomal regulators are selectively essential for toxicity of noncleavable linker ADCs. We further show that cleavable VC linkers are rapidly processed upon internalization and therefore surprisingly appear to bypass the requirement of lysosomal delivery. Lastly, we show that inhibition of sialic acid biosynthesis sensitizes cells to ADC treatment by increasing the rate of ADC internalization. This sensitization was observed using several ADCs targeting different antigens in diverse cancer cell types, including the FDA-approved ADC trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal novel regulators of endolysosomal trafficking, provide important insights to guide future ADC design, and identify candidate combination therapy targets as well as potential mechanisms of ADC resistance.

Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly-line polyketide synthase (PKS) that presumably synthesizes an unknown natural product. Here, we report the discovery and structure elucidation of the NOCAP (NOCardiosis-Associated Polyketide) aglycone by first fully reconstituting the NOCAP synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assemblyline PKS either in vitro or in E. coli, and of using these approaches to "deorphanize" a complete assembly-line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.

In response to biotic stress, plants reshape their complement of lipids to produce suites of highly modified fatty acids that bear unusual chemical functionality. Despite their chemical complexity, proposed roles in pathogen defense and presence in crop plants, little is known about the 25 biosynthesis of these decorated fatty acids. Falcarindiol is a prototypical member of a suite of acetylenic lipids from carrot, tomato, and celery that inhibits growth of several fungal strains and human cancer cell lines. Here we report a set of clustered genes in tomato (Solanum lycopersicum) that are required for the production of falcarindiol in leaves in response to treatment with an adapted fungal pathogen, Cladosporium fulvum. Our approach is based on 30 correlation of untargeted transcriptomic and metabolomic data sets in order to rapidly identify a candidate biosynthetic pathway. By reconstituting the initial biosynthetic steps in a heterologous host (Nicotiana benthamiana) and generating stable transgenic pathway mutants in tomato, we demonstrate a direct role for three genes in the cluster in falcarindiol biosynthesis. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens, and 35 provides critical insight into the biochemistry of alkynyl lipid production. One Sentence Summary:A biosynthetic gene cluster for the production of falcarindiol, a highly modified antifungal oxylipin found in edible plants. 40