No abstract

PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. We screened for small molecule inhibitors of the PRMT5–PBM interaction and validated a compound series which binds to the PRMT5–PBM interface and directly inhibits binding of SAPs. Mode of action studies revealed the formation of a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts PRMT5–RIOK1 complexes, and reduces substrate methylation. BRD0639 is a first-in-class PBM-competitive inhibitor that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions.

Activation of the nuclear factor (erythroid‐derived 2)‐like 2 (NFE2L2 or NRF2) transcription factor is a critical and evolutionarily conserved cellular response to oxidative stress, metabolic stress, and xenobiotic insult. Deficiency of NRF2 results in hypersensitivity to a variety of stressors, whereas its aberrant activation contributes to several cancer types, most commonly squamous cell carcinomas of the esophagus, oral cavity, bladder, and lung. Between 10% and 35% of patients with squamous cell carcinomas display hyperactive NRF2 signaling, harboring activating mutations and copy number amplifications of the NFE2L2 oncogene or inactivating mutations or deletions of KEAP1 or CUL3, the proteins of which co‐complex to ubiquitylate and degrade NRF2 protein. To better understand the role of NRF2 in tumorigenesis and more broadly in development, we engineered the endogenous Nfe2l2 genomic locus to create a conditional mutant LSL‐Nrf2E79Q mouse model. The E79Q mutation, one of the most commonly observed NRF2‐activating mutations in human squamous cancers, codes for a mutant protein that does not undergo KEAP1/CUL3‐dependent degradation, resulting in its constitutive activity. Expression of NRF2 E79Q protein in keratin 14 (KRT14)‐positive murine tissues resulted in hyperplasia of squamous cell tissues of the tongue, forestomach, and esophagus, a stunted body axis, decreased weight, and decreased visceral adipose depots. RNA‐seq profiling and follow‐up validation studies of cultured NRF2E79Q murine esophageal epithelial cells revealed known and novel NRF2‐regulated transcriptional programs, including genes associated with squamous cell carcinoma (e.g. Myc), lipid and cellular metabolism (Hk2, Ppard), and growth factors (Areg, Bmp6, Vegfa). These data suggest that in addition to decreasing adipogenesis, KRT14‐restricted NRF2 activation drives hyperplasia of the esophagus, forestomach, and tongue, but not formation of squamous cell carcinoma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PRMT5 is an arginine methyltransferase and a therapeutic target in MTAP null cancers. PRMT5 utilizes adaptor proteins for substrate recruitment through a previously undefined mechanism. Here, we identify an evolutionarily conserved peptide sequence shared among the three known substrate adaptors (pICln/CLNS1A, RIOK1 and COPR5) and show it is necessary and sufficient for interaction with PRMT5. We structurally resolve the interface with PRMT5 and show via genetic perturbation that it is required for methylation of adaptor-recruited substrates including the spliceosome, histones, and ribosome assembly complexes. Genetic disruption of the PRMT5-substrate adaptor interface leads to a hypomorphic decrease in growth of MTAP null tumor cells and is thus a novel site for development of therapeutic inhibitors of PRMT5.

PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. We screened for small molecule inhibitors of the PRMT5-PBM interaction and validated a compound series which binds to the PRMT5-PBM interface and directly inhibits binding of SAPs. Mode of action and structure determination studies revealed that these compounds form a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts the PRMT5-RIOK1 complex, and reduces substrate methylation. BRD0639 is a first-in-class PBM-competitive small molecule that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions.