The Arp2/3 complex is an actin nucleator with well-characterized activities in cell morphogenesis and movement, but its roles in nuclear processes are relatively understudied. We investigated how the Arp2/3 complex affects genomic integrity and cell cycle progression using mouse fibroblasts containing an inducible knockout (iKO) of the ArpC2 subunit. We show that permanent Arp2/3 complex ablation results in DNA damage, the formation of cytosolic micronuclei, and cellular senescence. Micronuclei arise in ArpC2 iKO cells due to chromatin segregation defects during mitosis and premature mitotic exits. Such phenotypes are explained by the presence of damaged DNA fragments that fail to attach to the mitotic spindle, abnormalities in actin assembly during metaphase, and asymmetric microtubule architecture during anaphase. In the nuclei of Arp2/3-depleted cells, the tumor suppressor p53 is activated and the cell cycle inhibitor Cdkn1a/p21 mediates a G1 arrest. In the cytosol, micronuclei are recognized by the DNA sensor cGAS, which is important for stimulating a STING- and IRF3-associated interferon response. These studies establish functional requirements for the mammalian Arp2/3 complex in mitotic spindle organization and genome stability. They also expand our understanding of the mechanisms leading to senescence and suggest that cytoskeletal dysfunction is an underlying factor in biological aging.

The Arp2/3 complex is a ubiquitous actin nucleator with well-characterized activities in cell organization and movement, but its roles in chromatin-associated and cell cycle-related processes are relatively understudied. We investigated how the Arp2/3 complex affects genomic integrity, mitosis, and cell proliferation using mouse fibroblasts containing an inducible knockout (iKO) of the ArpC2 subunit. We show that permanent Arp2/3 ablation results in DNA damage, the formation of cytosolic micronuclei, and cellular senescence. Upon Arp2/3 depletion, cells undergo an abrupt proliferation arrest that is accompanied by activation of the tumor suppressor p53, upregulation of its downstream cell cycle inhibitor Cdkn1a/p21, and recognition of micronuclei by the cytosolic DNA sensor cGAS. Micronuclei arise in ArpC2 iKO cells due to chromosome segregation defects during mitosis and premature mitotic exits. Such phenotypes are explained by the presence of damaged chromatin fragments that fail to attach to the mitotic spindle, abnormalities in actin assembly during metaphase, and asymmetric microtubule architecture during anaphase. These studies establish functional requirements for the mammalian Arp2/3 complex in genome stability and mitotic spindle organization. They further expand our understanding of the intracellular mechanisms that lead to senescence and suggest that cytoskeletal dysfunction is an underlying factor in biological aging.

Pharmacological inhibitors of TEAD transcription factors have emerged as a promising novel class of anti-cancer agents. TEAD inhibitors disrupt oncogenic YAP/TAZ signaling, resulting in cell cycle arrest and cell death in susceptible cancers. In preclinical models, cancer cell lines with alterations in the Hippo signaling pathway, such as mutations or loss of NF2 or LATS, are particularly sensitive to inhibition of TEAD signaling. In addition, YAP/TAZ-TEAD signaling is thought to play a role in resistance to several targeted therapies, such as EGFR inhibitors and MEK inhibitors. BGI-9004 is a covalent inhibitor of TEAD1-4 with low nanomolar potency in a TEAD reporter cell line and anti-cancer activity in vitro and in vivo. In the NF2-deficient NCI-H226 and the NF2-wildtype, LATS-mutant MST-O211H mesothelioma xenograft models, BGI-9004 treatment resulted in sustained tumor regressions over a 28d treatment course. Anti-cancer activity was accompanied by significant inhibition of TEAD-mediated transcription in BGI-9004-treated tumors, and no adverse effects on the body weight of treated mice were observed. Moreover, proteome-wide protein binding of BGI-9004 was assessed by Isobaric Mass Tagged Affinity Characterization (IMTACTM), which confirmed its high selectivity for TEAD. In line with this, BGI-9004 had a clean profile in a panel of protein targets that present potential safety liabilities (CEREP safety panel). These findings prompted us to explore the combination potential of BGI-9004 with other targeted therapies, including inhibitors of EGFR, MEK and mutant KRASG12C. Here, we found that BGI-9004 potentiated the effect of other targeted therapies. In particular, we generated dose response matrices for BGI-9004 and inhibitors of KRASG12C and KRASG12D in several KRAS-mutant cell lines, including three lung cancer cell lines, and demonstrated synergy for the combination. These results suggest that inhibition of YAP/TAZ-TEAD signaling may improve the efficacy of KRAS-targeted therapy. Taken together, the covalent TEAD inhibitor BGI-9004 has demonstrated promising activity both as a single agent and in combination with other targeted agents, a favorable pharmacokinetic profile and high target selectivity in preclinical models, supporting its evaluation as a novel anti-cancer agent in clinical trials. Citation Format: Shirley Guo, Shashank Shrishrimal, Jason Cui, Guoqing Wang, Vivian Zhang, Ning Deng, Iris Dong, Anna Chen, Steve Luo, David Sperandio, Ping Cao, Wolf R. Wiedemeyer. Preclinical characterization of BGI-9004, a covalent TEAD inhibitor with exceptional anti-cancer activity and combination potential. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4976.

WDR5, the histone H3 lysine 4 (H3K4) presenter WD repeat-containing protein 5, is a widely expressed protein that forms complexes with H3K4 methyltransferases MLL1-MLL4 and partner proteins including RBBP5, ASH2L, DPY30, and MYC. It plays an important role in epigenetic machinery assembly, transcriptional regulation, and chromatin regulation. As a result, WDR5 has become an increasingly attractive target for therapeutic intervention against cancers, including mixed-lineage leukemia, neuroblastoma, breast cancer, bladder cancer, pancreatic cancer, and colorectal cancer. Using its proprietary chemoproteomic platform IMTACTM (Isobaric Mass Tagged Affinity Characterization), BridGene has screened its unique covalent library against live-cell proteomes and discovered small-molecule ligands for hundreds of hard-to-drug targets, including transcription factors, splicing factors, epigenetic modulators, and E3 ligases. WDR5 is among these targets that BridGene has discovered a novel ligand for. Preliminary characterization of the “hit” compound, BGS1989, showed that it interacts with WDR5 in a dose-dependent manner, and it can inhibit MLL1 methyltransferase activity at low µM potency. After one round of chemical optimization, we identified an improved inhibitor BGS2597 that has ~200 nM potency in the WDR5-MLL interaction and H3K4 methylation assays. Additional optimizations are being conducted to further improve BGS2597’s activity and selectivity for WDR5. Comprehensive characterizations are being performed to delineate which of WDR5’s functions are modulated by BGS2597. The WDR5 inhibitor discovered using IMTACTM platform provides an excellent starting point for the development of new drugs targeting WDR5-dependent cancers. Citation Format: Cindy Huang, Shirley Guo, Ping Cao. Discovery of novel small-molecule inhibitors for an epigenetic modulator WDR5 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P089.