The Werner protein, WRN, is a member of the RecQ helicase family implicated in genome maintenance. Several large-scale functional genomics screens have identified WRN as a synthetic lethal target in cancer cell lines with microsatellite instability-high (MSI-H). Accordingly, WRN is considered a potential therapeutic target in MSI-H cancers. HRO761, a non-covalent WRN inhibitor developed by Novartis, entered clinical trial for patients with MSI-H colorectal cancer (CRC). In this study, we investigated bioisosteric replacement of the hydroxyl pyrimidine residue of HRO761 with several bicyclic structures to obtain a novel chemical entity. In vitro ATPase and cell proliferation assays revealed two candidate chemicals that showed similar or better effects than HRO761. Additionally, an in vivo study demonstrated that KWR095, a newly synthesized WRN inhibitor, has significant anti-proliferative effects compared with vehicle.

Targeting undruggable proteins by inducing proximity between E3 ligase and their substrates has emerged as an innovative strategy for tackling challenging diseases. In this study, we identified a novel GSPT1 degrader, 4a (KMG-1068), through screening of our in-house small molecule library. Treatment with 4a demonstrated significant anti-proliferative activity across multiple cell lines, which was diminished by co-treatment with MLN4924, suggesting the involvement of the Cullin-containing complex. Quantitative proteomic analysis indicated that 4a predominantly induces the degradation of GSPT1/2. We further validated that 4a-mediated GSPT1/2 degradation is dependent on both CUL4 and CRBN. Moreover, 4a forms a ternary complex with CRBN and GSPT1/2, albeit with weaker binding affinity compared to reported GSPT1 molecular glues. BRET assays and competition assays with pomalidomide demonstrated that 4a binds to the C-terminal IMiD binding site of CRBN, leading to the degradation of GSPT1. Despite lacking the characteristic glutarimide moiety present in other CRBN-based molecular glue degraders, 4a interacts effectively with the IMiD binding site of CRBN. Structural characterization through analog synthesis further underscored the importance of specific structural features for CRBN engagement, GSPT1/2 degradation, and anti-proliferative effects, establishing 4a as a promising novel GSPT1/2 degrader with significant therapeutic potential.

Targeted protein degradation has emerged as a novel therapeutic paradigm in drug discovery. Despite the FDA approval of anaplastic lymphoma kinase (ALK) inhibitors, the pursuit of compounds with enhanced potency and prolonged efficacy remains crucial to mitigate inevitable adverse effects. In this context, we endeavored to develop ALK degraders utilizing FDA-approved ALK inhibitors-crizotinib, ceritinib, brigatinib, and alectinib-as ALK binders, along with 4-methoxyphenylfumarate as a covalent handle to bind to RNF126 E3 ligase. Among the synthesized compounds, dALK-3-derived from brigatinib-efficiently induced the proteasomal degradation of EML4-ALK and exhibited a 10-fold superior anti-proliferative effect on H3122 cells compared to brigatinib. However, the enhanced anti-proliferative activity of dALK-3 was found to be independent of RNF126, a presumed potential E3 ligase, suggesting the need for investigation of other components within the ubiquitin-proteasome system. Our findings further support the potential application of the fumarate moiety as a binder for E3 ligases in targeted protein degradation.

The interplay between multiple transcription factors precisely regulates eukaryotic transcription. Here, we report that the protein methyltransferases, MLL2/KMT2B and PRMT1, interact directly and act collectively to regulate gene expression. PRMT1 binds to the N-terminal region of MLL2, considered an intrinsically disordered region, and methylates multiple arginine residues within its RGG/RG motifs. Notably, overexpression of PRMT1 decreased poly-ubiquitylation of MLL2, whereas mutations on methylation sites in MLL2 increased MLL2 poly-ubiquitylation, suggesting that PRMT1-mediated methylation stabilizes MLL2. MLL2 and PRMT1 cooperatively stimulated the expression of a chromosomal reporter gene in a PRMT1-mediated, MLL2-methylation-dependent manner. RNA-seq analysis found that MLL2 and PRMT1 jointly regulate the expression of genes involved in cell membrane and extracellular matrix functions, and depletion of either resulted in impaired cell migration and invasion. Our study provides evidence that PRMT1-mediated MLL2 methylation regulates MLL2 protein stability and the expression of their target genes.

Abstract The integration of big data analytics with cancer research is catalyzing a transformative approach in cancer treatment, primarily focusing on the discovery of novel and efficacious anticancer targets. Our study presents an advanced algorithm, specifically crafted to exploit the extensive data available in the field of oncology. We started with the genomic and clinical information of 8,864 patients with 33 different cancers (TCGA). Then we implemented the following algorithm to discover anti-cancer targets by analyzing the clinical significance (cBioPortal), drug development status (Cortellis), and oncogenicity (DepMap) of candidate genes: Candidate genes = {gene | gene ∈ Genes, [Frequency(gene) > 50, Drug(gene) ∈ {'biological testing', 'preclinical stage'}, Association(gene) ≥ 0.4, Publication(gene) ≤ 200] ∨ [Publication(gene) ≥ 200 ∧ Boolean(gene)]}. We employed this algorithm to analyze fusion genes, which represent promising anti-cancer targets known for their potential to elicit substantial clinical responses, but there is a high demand for new ones. We identified four druggable therapeutic targets out of a total of 15,291 fusion genes through the algorithm: frame-shifted FGFR3-TACC3, in-framed DLK1-RPS11, frame-shifted CHP1-RAD51B, and in-framed TBC1D22A-SMYD3. We conducted in vitro validation studies of these fusion genes in NIH3T3 cell lines, and it confirmed that all of the fusion genes not only produce mRNA and protein levels but also induce oncogenic effects on cellular behavior. In the case of FGFR3-TACC3, the introduced fusion gene induced mRNA (p < 0.05) and protein expression (p < 0.05) even when frame-shifted. In addition, the proliferation rate of transformed cells increased more than 4-fold on day 10 (p < 0.0001) and colony formation increased more than 5-fold on day 21 (p < 0.01) compared to wild-type cells. These results demonstrate the tumorigenicity of the fusion genes. Taken together, this study emphasizes the crucial role of big data in propelling oncology research forward. The algorithm we developed can offer a new pathway for creating innovative cancer treatment, marking a significant advancement in the realm of personalized cancer therapy. Citation Format: Dooho Kim, Jong Woo Park, Jung-Ae Kim, Jeong-Hoon Kim, Tae Sub Park, Joonghoon Park. Big data-driven discovery of novel oncogenic fusion genes for anticancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5954.

AbstractHistone lysine methylation is pivotal in shaping the epigenetic landscape and is linked to cell physiology. Coordination of the activities of multiple histone lysine methylation modifiers, namely, methyltransferases and demethylases, modulates chromatin structure and dynamically alters the epigenetic landscape, orchestrating almost all DNA-templated processes, such as transcription, DNA replication, and DNA repair. The stability of modifier proteins, which is regulated by protein degradation, is crucial for their activity. Here, we review the current knowledge of modifier-protein degradation via specific pathways and its subsequent impact on cell physiology through epigenetic changes. By summarizing the functional links between the aberrant stability of modifier proteins and human diseases and highlighting efforts to target protein stability for therapeutic purposes, we aim to promote interest in defining novel pathways that regulate the degradation of modifiers and ultimately increase the potential for the development of novel therapeutic strategies.

Effects of BJ-1301 and α-TOH on cell viability, proliferation, and VEGF-induced tube formation. A, Effects of α-TOH or BJ-1301 on the viability of HUVECs and BAOECs were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. B, Effect of BJ-1301 alone treatment on HUVEC proliferation. C, HUVECs plated in a well coated with Matrigel basement membrane matrix, were treated with compounds in the presence of VEGF (20 ng/ml). The total tube length per field was measured using Image Inside software (Upper panel). Invasion of HUVECs was measured using VEGF-induced HUVEC migration in the Transwell assay (Lower panel). The bar graphs represent the mean {plus minus} S.E.M. of three independent experiments. *P<0.05 versus vehicle-treated control. #P<0.05 versus VEGF alone. D, Inhibitory effect of BJ-1301 on VEGF-induced tube formation in a time-dependent manner.

Von Hippel-Lindau (VHL) is a tumor suppressor that functions as the substrate recognition subunit of the CRL2VHL E3 complex. While substrates of VHL have been identified, its tumor suppressive role remains to be fully understood. For further determination of VHL substrates, we analyzed the physical interactome of VHL and identified the histone H3K9 methyltransferase SETBD1 as a novel target. SETDB1 undergoes oxygen-dependent hydroxylation by prolyl hydroxylase domain proteins and the CRL2VHL complex recognizes hydroxylated SETDB1 for ubiquitin-mediated degradation. Under hypoxic conditions, SETDB1 accumulates by escaping CRL2VHL activity. Loss of SETDB1 in hypoxia compared with that in normoxia escalates the production of transposable element-derived double-stranded RNAs, thereby hyperactivating the immune-inflammatory response. In addition, strong derepression of TEs in hypoxic cells lacking SETDB1 triggers DNA damage-induced death. Our collective results support a molecular mechanism of oxygen-dependent SETDB1 degradation by the CRL2VHL E3 complex and reveal a role of SETDB1 in genome stability under hypoxia.

Abstract Background Lysosomes are closely linked to autophagic activity, which plays a vital role in pancreatic ductal adenocarcinoma (PDAC) biology. The survival of PDAC patients is still poor, and the identification of novel genetic factors for prognosis and treatment is highly required to prevent PDAC-related deaths. This study investigated the germline variants related to lysosomal dysfunction in patients with PDAC and to analyze whether they contribute to the development of PDAC. Methods The germline putative pathogenic variants (PPV) in genes involved in lysosomal storage disease (LSD) was compared between patients with PDAC (n = 418) and healthy controls (n = 845) using targeted panel and whole-exome sequencing. Furthermore, pancreatic organoids from wild-type and Kras G12D mice were used to evaluate the effect of lysosomal dysfunction on PDAC development. RNA sequencing (RNA-seq) analysis was performed with established PDAC patient-derived organoids (PDOs) according to the PPV status. Results The PPV in LSD-related genes was higher in patients with PDAC than in healthy controls (8.13 vs. 4.26%, Log2 OR = 1.65, P = 3.08 × 10–3). The PPV carriers of LSD-related genes with PDAC were significantly younger than the non-carriers (mean age 61.5 vs. 65.3 years, P = 0.031). We further studied a variant of the lysosomal enzyme, galactosylceramidase (GALC), which was the most frequently detected LSD variant in our cohort. Autophagolysosomal activity was hampered when GALC was downregulated, which was accompanied by paradoxically elevated autophagic flux. Furthermore, the number of proliferating Ki-67+ cells increased significantly in pancreatic organoids derived from G alc knockout Kras G12D mice. Moreover, GALC PPV carriers tended to show drug resistance in both PDAC cell line and PDAC PDO, and RNA-seq analysis revealed that various metabolism and gene repair pathways were upregulated in PDAC PDOs harboring a GALC variant. Conclusions Genetically defined lysosomal dysfunction is frequently observed in patients with young-onset PDAC. This might contribute to PDAC development by altering metabolism and impairing autophagolysosomal activity, which could be potentially implicated in therapeutic applications for PDAC.

ABSTRACT Lysosome is closely linked to autophagy, which plays a vital role in pancreatic adenocarcinoma (PDAC) tumor biology. This study investigated whether lysosome storage dysfunction (LSD) contributes to PDAC development. Germline putative pathogenic variants (PPVs) in genes involved in lysosome functions were compared between PDAC patients (N=418) and healthy controls (N=845). Furthermore, Galc -knockout mouse pancreas organoids and human PDAC organoids were used to evaluate the consequences of PPV status in PDAC development and establishment. LSD PPVs were enriched in PDAC patients (Log2OR=1.65, P=3.08×10 −3 ). PPV carriers diagnosed with PDAC were younger than non-carriers (61.5 vs. 65.3 years, P=0.031). Hampered autophagolysosome activity with increased autophagy flux and elevated Ki-67 index were observed following GALC downregulation. RNA sequencing of human PDAC organoids revealed upregulation of metabolism related to LSD. Genetically defined lysosome dysfunction is frequently observed in young-age onset PDACs. Lysosome dysfunction might contribute to PDAC development via altered metabolism and impaired autophagolysosome activity.

Differential expression of NOX homologs in A549 and H1299 human lung cancer cells. A, Quantitative analysis of mRNA extracted from the two cancer cell lines and positive control cell lines was performed using ABI PCR primers and Quantitect PCR probe mix. Caco2 and HT29 cell lines were used for NOX1, DU145 for NOX2, p67phox and p47phox, and HUVEC for NOX4. *P<0.05 versus positive control group. #P<0.05 versus A549 cells. B, mRNA copy number of NOX1, NOX2 and NOX4 was determined in A549 and H1299 lung cancer cells.

Inhibitors of apoptosis proteins (IAPs), defined by the presence of baculovirus IAP repeat (BIR) protein domain, are critical regulators of cell survival and cell death processes. Cellular IAP 1/2 (cIAP1/2) and X-linked IAPs (XIAPs) regulate the innate immune signaling pathway through their E3 ubiquitin ligase activity. Peptidomimetics or small-molecule IAP antagonists have been developed to treat various diseases, such as cancer, infection, and inflammation. In this study, we synthesized and characterized IAP-cereblon (CRBN) heterodimerizing proteolysis-targeting chimera (PROTAC), which induces the degradation of cIAP1/2 and XIAP but not CRBN. We demonstrated that this PROTAC inhibits tumor necrosis factor alpha (TNFα)-induced innate immune response and cancer cell migration and invasion, leading to apoptotic cell death. Our study is the first to demonstrate that both cIAPs and XIAP are degradable when applied to the PROTAC strategy.

BACKGROUND:Elevated expression of Dickkopf-1 (DKK1) is frequently observed in hepatocellular carcinoma (HCC) patients with poor clinical outcomes. Several reports indicating the functional involvement of DKK1 in HCC progression have suggested DKK1 as a promising therapeutic target for HCC.OBJECTIVE:In this study, to develop an efficient way to target DKK1, we assessed the effect of CDK9 inhibitors on DKK1 expression linked to metastatic movement of HCC.METHODS:The expression of DKK1 in CDK9 inhibitor-treated HCC cells was measured by western blot, ELISA and quantitative real-time reverse transcription PCR. Wound healing assay, migration assay, invasion assay and western blot were examined to evaluate the functional role of DKK1 in CDK9 inhibitors-treated HCC.RESULTS:Inactivation of CDK9 either by a catalytic inhibitor being clinically evaluated or by a specific CDK9 protein degrader largely downregulated DKK1 expression at the transcript and protein levels. In addition, CDK9 inhibitors suppressed the migration and invasion of HCC cells. We observed that ectopic high expression of DKK1 at least partially reversed the defects in metastatic movement of HCC cells mediated by CDK9 inhibitors. We further discovered that the DKK1-nuclear β-catenin axis associated with the metastatic potential of HCC cells was impaired by CDK9 inhibitors.CONCLUSION:Taken together, our findings suggest that CDK9 inhibitors are potent tools to target DKK1, which can suppress the metastatic progression of HCC.

Hepatocellular carcinoma (HCC) is an aggressive and incurable cancer. Although understanding of the molecular pathogenesis of HCC has greatly advanced, therapeutic options for the disease remain limited. In this study, we demonstrated that SETD5 expression is positively associated with poor prognosis of HCC and that SETD5 depletion decreased HCC cell proliferation and invasion while inducing cell death. Transcriptome analysis revealed that SETD5 loss downregulated the interferon-mediated inflammatory response in HCC cells. In addition, SETD5 depletion downregulated the expression of a critical glycolysis gene, PKM (pyruvate kinase M1/2), and decreased glycolysis activity in HCC cells. Finally, SETD5 knockdown inhibited tumor growth in xenograft mouse models. These results collectively suggest that SETD5 is involved in the tumorigenic features of HCC cells and that targeting SETD5 may suppress HCC progression.

AIMS Cereblon (CRBN) is a substrate receptor of the E3 ubiquitin ligase complex that was reported to target ion channel proteins. L-type voltage-dependent Ca2+ channel (LTCC) density and dysfunction is a critical player in heart failure with reduced ejection fraction (HFrEF). However, the underlying cellular mechanisms by which CRBN regulates LTCC subtype Cav1.2α during cardiac dysfunction remain unclear. Here, we explored the role of CRBN in HFrEF by investigating the direct regulatory role of CRBN in Cav1.2α activity and examining how it can serve as a target to address myocardial dysfunction. METHODS AND RESULTS Cardiac tissues from HFrEF patients exhibited increased levels of CRBN compared with controls. In vivo and ex vivo studies demonstrated that whole-body CRBN knockout (CRBN-/-) and cardiac-specific knockout mice (Crbnfl/fl/Myh6Cre+) exhibited enhanced cardiac contractility with increased LTCC current (ICaL) compared with their respective controls, which was modulated by the direct interaction of CRBN with Cav1.2α. Mechanistically, the Lon domain of CRBN directly interacted with the N-terminal of Cav1.2α. Increasing CRBN levels enhanced the ubiquitination and proteasomal degradation of Cav1.2α and decreased ICaL. In contrast, genetic or pharmacological depletion of CRBN via TD-165, a novel PROTAC-based CRBN degrader, increased surface expression of Cav1.2α and enhanced ICaL. Low CRBN levels protected the heart against cardiomyopathy in vivo. CONCLUSION Cereblon selectively degrades Cav1.2α, which in turn facilitates cardiac dysfunction. A targeted approach or an efficient method of reducing CRBN levels could serve as a promising strategy for HFrEF therapeutics. KEY QUESTION KEY FINDING TAKE-HOME MESSAGE Cereblon modulates cardiac function by altering Cav1.2α current density and CRBN-targeting therapy could serve as a novel strategy for future HFrEF therapeutics.

On the basis of the previously reported polypharmacological profile of truncated d-1'-homologated adenosine derivatives [J. Med. Chem.2020, 63, 16012], the l-nucleoside analogues were synthesized using computer-aided design and evaluated for biological activity. The target molecules were synthesized from d-ribose via the key intramolecular cyclization of the monotosylate and Mitsunobu condensation. The peroxisome proliferator-activated receptor (PPAR) binding activities of l-nucleoside analogue 2d (Ki = 4.3 μM for PPARγ and 1.0 μM for PPARδ) were significantly improved in comparison with those of the d-nucleoside compound 1 (11.9 and 2.7 μM, respectively). In addition, the l-nucleosides showed more potent adiponectin-secretion-promoting activity than the d-nucleoside analogues.

Advanced hepatocellular carcinoma (HCC) is among the most challenging cancers to overcome, and there is a need for better therapeutic strategies. Among the different cancer drugs that have been used in clinics, sorafenib is considered the standard first-line drug for advanced HCC. Here, to identify a chemical compound displaying a synergistic effect with sorafenib in HCC, we screened a focused chemical library and found that MG149, a histone acetyltransferase inhibitor targeting the MYST family, exhibited the most synergistic anticancer effect with sorafenib on HCC cells. The combination of sorafenib and MG149 exerted a synergistic anti-proliferation effect on HCC cells by inducing apoptotic cell death. We revealed that cotreatment with sorafenib and MG149 aggravated endoplasmic reticulum (ER) stress to promote the death of HCC cells rather than adaptive cell survival. In addition, combined treatment with sorafenib and MG149 significantly increased the intracellular levels of unfolded proteins and reactive oxygen species, which upregulated ER stress. Collectively, these results suggest that MG149 has the potential to improve the efficacy of sorafenib in advanced HCC via the upregulation of cytotoxic ER stress.

Cereblon (CRBN) is a multi-functional protein that acts as a substrate receptor of the E3 ligase complex and a molecular chaperone. While CRBN is proposed to function in mitochondria, its specific roles are yet to be established. Here, we showed that knockdown of CRBN triggers oxidative stress and calcium overload in mitochondria, leading to disruption of mitochondrial membrane potential. Notably, long-term CRBN depletion using PROteolysis TArgeting Chimera (PROTAC) induced irreversible mitochondrial dysfunction, resulting in cell death. Our collective findings indicate that CRBN is required for mitochondrial homeostasis in cells. [BMB Reports 2021; 54(6): 305-310].