The development of drug resistance is a major cause of poor prognosis and therapeutic failure in NSCLC. The mutual synergism between cMET and EGFR is known to promote acquired drug resistance, which complicates effective treatment strategies. In NSCLC, cMET gene amplification is reported in 2-4% of previously untreated patients and in 5-20% of patients with EGFR mutations and acquired resistance to EGFR TKIs. This crosstalk between the cMET and EGFR pathways underscores the need for dual inhibition as a promising treatment for NSCLC patients, especially those with acquired cMET activation. cMET, a proto-oncogene encoding the RTK HGFR, plays a crucial role in cell proliferation, motility, and survival. Aberrant activation of cMET through gene amplification, mutation, or overexpression has been implicated in the development of various cancers, including NSCLC. EGFR, another critical RTK, is frequently mutated in NSCLC, leading to uncontrolled cell growth and survival. The interplay between cMET and EGFR signaling pathways facilitates the emergence of resistance to EGFR TKIs, a common occurrence in NSCLC patients undergoing treatment. This resistance often results from secondary mutations in the EGFR gene, such as T790M and C797S, or from activation of alternative signaling pathways, including cMET amplification. CKD-702 is a bispecific antibody specifically designed to target both cMET and EGFR. It is a tetravalent IgG1-like bispecific antibody, in which a single-chain variable fragment specific to EGFR is genetically fused to the C-terminus of a conventional IgG1 of cMET. This innovative design allows CKD-702 to bind with high affinity to the extracellular domains of both cMET and EGFR, effectively inhibiting their signaling by blocking ligand binding. Additionally, CKD-702 induces the internalization and degradation of both receptors, thereby further inhibiting their activity and downstream signaling pathways. This study aims to evaluate the efficacy of combination therapy with EGFR TKIs and CKD-702 for overcoming resistance in NSCLC, particularly in EGFR exon 20 insertion mutant models and triple mutant models, including the C797S mutation. The combination therapy demonstrates superior efficacy compared to monotherapy in both cellular and animal models, effectively inhibiting signaling pathways and tumor growth. These findings are promising for addressing the significant challenge posed by the development of resistance to EGFR TKIs in the treatment of NSCLC. A Phase I study of CKD-702, which began in 2020, is currently ongoing. Current clinical trials of CKD-702 monotherapy show promising results, with combination therapy being considered to address resistance mechanisms in NSCLC. Mi Jin Yoon, Soon-Ki Hong, Gun-Woo Park, Kyung-Woo Lee, Sunhong Kim, Young-Shin Kwak, Ju-Hee Lee. CKD-702, a novel bispecific antibody, enhances anti-tumor effect of combination with EGFR TKIs in NSCLC preclinical model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1708.

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.

Chronic exposure to stress or unwanted stimuli has been known to activate kappa opioid receptor/dynorphin (KOR/DYN) systems, which could induce depressive states and develop into some psychiatric disorders. Here, we report the first discovery of pyrazoloisoquinoline-based novel KOR β-arrestin inverse agonists through synthesis, structure-activity relationships, optimization, and the biological evaluations of μ/κ/δ opioid receptor activities with cAMP and β-arrestin recruitment assays. The optimized compound 7q shows potent and selective β-arrestin inverse agonism at KOR with an EC50 value of 9.33 nM in contrast to lower activities at DOR and no activity at MOR. Moreover, we use molecular dynamics simulations to predict the binding mode of the inverse agonist and propose a mechanism for the inverse agonism. We find that the transmembrane helix 6 position of the activated state is different for the OR subtypes, leading to significantly different interactions between the receptor and β-arrestin.

GPR43 (also known as FFAR2), a metabolite-sensing G-protein-coupled receptor stimulated by short-chain fatty acid (SCFA) ligands is involved in innate immunity and metabolism. GPR43 couples with Gαi/o and Gαq/11 heterotrimeric proteins and is capable of decreasing cyclic AMP and inducing Ca2+ flux. The GPR43 receptor has additionally been shown to bind β-arrestin 2 and inhibit inflammatory pathways, such as NF-ΚB. However, GPR43 shares the same ligands as GPR41, including acetate, propionate, and butyrate, and determination of its precise functions in association with endogenous ligands, such as SCFAs alone, therefore remains a considerable challenge. In this study, we generated novel synthetic agonists that display allosteric modulatory effects on GPR43 and downregulate NF-ΚB activity. In particular, the potency of compound 187 was significantly superior to that of preexisting compounds in vitro. However, in the colitis model in vivo, compound 110 induced more potent attenuation of inflammation. These novel allosteric agonists of GPR43 clearly display anti-inflammatory potential, supporting their clinical utility as therapeutic drugs.

μ‐Opioid receptor (MOR) Gi‐biased agonists with no recruitment of β‐arrestin were introduced as a new analgesic strategy to overcome the conventional undesirable side effects of opioid receptor‐targeted drugs, such as tolerance, addiction, respiratory depression, and constipation. For the development of novel Gi‐biased MOR agonists, the design, synthesis, and structure–activity relationship (SAR) analysis of the aminopyrazole core skeleton were conducted according to the current SAR data of PZM21 (2a) and its derivatives. New derivatives were biologically evaluated for their agonistic effects on cyclic adenosine monophosphate (cAMP) levels for the Gi pathway and β‐arrestin recruitment in MOR/κ‐opioid receptor/δ opioid receptor. An optimized selective Gi‐biased agonist, Compound 17a, was discovered with potent cAMP inhibitory activities, with a 50% efficacy concentration value of 87.1 nM and no activity in the MOR β‐arrestin pathway and other subtypes. The in vivo pain relief efficacy of Compound 17a was confirmed in a dose‐dependent manner with spinal nerve ligation and cisplatin‐induced peripheral neuropathy rodent neuropathic pain models.

Endothelin receptor A (EDNRA) has been reported to play various crucial physiological roles and has been shown to be associated with the pathology of several diseases, including colorectal cancer (CRC). However, the molecular mechanisms of EDNRA in the development of human CRC have not been fully elucidated to date. In this context, the present study was performed to investigate biological functions and novel downstream signaling pathways affected by EDNRA, during CRC progression. First, using public data repositories, it was observed that the EDRNA expression levels were markedly increased in CRC tissues, as compared to normal tissues. Patients with CRC with an increased EDNRA expression exhibited a significantly decreased survival rate in comparison with those with a lower EDNRA expression. Furthermore, a positive correlation between the levels of EDNRA and its ligand, EDN1, was found in CRC tissues. The ectopic expression of EDNRA or its ligand, EDN1, promoted, whereas the silencing of EDNRA or EDN1 decreased cell proliferation and migration in vitro. To elucidate the signaling pathways involved in the regulation of EDNRA expression in CRC cells, a phosphokinase array analysis was performed, and it was observed that the knockdown of EDNRA substantially suppressed the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in CRC cells. Of note, STAT3 silencing simultaneously decreased EDN1 and EDNRA expression, with the expression of EDN1 and/or EDNRA appearing to be directly regulated by binding STAT3 to their promoter region, according to chromatin immunoprecipitation and promoter assays, ultimately indicating a positive feedback loop in the expression of EDNRA and EDN1. It was also observed that treatment with an EDNRA antagonist (macitentan), alone or in combination with cisplatin, suppressed cell growth and migration ability, and induced cell apoptosis. Collectively, these data suggest a critical role of the EDN1/EDNRA signaling pathway in CRC progression. Thus, the pharmacological intervention of this signaling pathway may prove to be a potential therapeutic approach for patients with CRC.

RON is a receptor tyrosine kinase of the MET proto-oncogene family, known as macrophage stimulating 1 receptor (MST1R) that recognizes macrophage-stimulating protein (MSP). RON has been known to be highly expressed in several epithelial tumors, promoting tumor progression and metastasis, and on macrophages surface, facilitating M2-like phenotype polarization of tumor-associated macrophages (TAM) to impair anti-tumor functions of CD8+ T cells. Although pharmacological inhibition of RON kinase has received attention from many research groups, monoclonal antibody drug showed a limited clinical efficacy due to RON variant of receptor region. Because the structure homology of kinase domain between RON and MET is 80%, discovery of RON-specific small molecule inhibitor is considered to be highly challenging to achieve. At first, we discovered a lead compound with about 50-fold of RON selectivity against MET kinase. Our medicinal chemistry efforts elevated RON/MET selectivity up to 500~10,000-fold (IC50 = 1~50 nM in RON enzyme assay), and the target specificity was proved through a panel assay of 372 kinases (Reaction Biology Corp.). A series of compounds effectively modulated p-RON and downstream signals (e.g. p-ERK and p-AKT) in MDA-MB-453, and inhibited cell proliferation of Ba/F3 cell-line engineered with human RON kinase. Significant anti-tumor effects were shown in syngeneic mouse models compared to a control group after oral administration, and studies related to immuno-oncology are ongoing in in-vitro or ex-/in-vivo assay systems. In conclusion, we have identified RON selective small molecule inhibitors that will be useful to figure out the biology following RON selective inhibition against MET kinase, and to validate the translational potential of RON kinase as a therapeutic target for human diseases including cancer. Citation Format: Young Jin Ham, GyeongHwan Kim, Juhyun Lee, Jaeyoung Lee, Min Kyung Kim, Sunhong Kim, Woosook Kim, Han Byoul Kim, Sung Woong Jang, Hee Dong Park, Seonguk Jeon, Seok-Joo Kim, Joongheui Cho, Jungjoon Kim, Hong bin Yoon, Youngshin Kwak. Discovery of highly selective RON kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB524.

LIN28 protein and let-7 family micro RNAs (miRNAs) that are an evolutionarily conserved from nematodes to humans are the important regulators of developmental timing by dynamically interacting with each other. However, regulators of LIN28 remain largely elusive. Here, we show the evidences that Sjögren Syndrome antigen B (SSB) protein associates and cooperates with LIN28A and LIN28B, mammalian orthologues of Caenorhabditis elegans lin-28, proteins in the nucleus. Knockdown of SSB in HEK293 cell line resulted in the decrease of the amount of LIN28B mRNAs and proteins, and the increase of the level of mature let-7 miRNAs. Furthermore, RNA interference of ssb-1 gene, a worm SSB orthologue, was sufficient to cause a heterochronic defect in seam cells of C. elegans, recapitulating the phenotype of lin-28 downregulation. Collectively, we suggest that SSB is an important regulator for the LIN28-let-7 axis.

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].

RalBP1 associated EPS domain containing 1 (REPS1) is conserved from Drosophila to humans and implicated in the endocytic system. However, an exact role of REPS1 remains largely unknown. Here, we demonstrated that mitogen activated protein kinase kinase (MEK)-p90 ribosomal S6 Kinase (RSK) signaling pathway directly phosphorylated REPS1 at Ser709 upon stimulation by epidermal growth factor (EGF) and amino acid. While REPS2 is known to be involved in the endocytosis of EGF receptor (EGFR), REPS1 knockout (KO) cells did not show any defect in the endocytosis of EGFR. However, in the REPS1 KO cells and the KO cells reconstituted with a non-phosphorylatable REPS1 (REPS1 S709A), the recycling of transferrin receptor (TfR) was attenuated compared to the cells reconstituted with wild type REPS1. Collectively, we suggested that the phosphorylation of REPS1 at S709 by RSK may have a role of the trafficking of TfR.

GPR43 (also known as FFAR2 or FFA2) is a G-protein-coupled receptor primarily expressed in immune cells, enteroendocrine cells and adipocytes that recognizes short-chain fatty acids, such as acetate, propionate, and butyrate, likely to be implicated in innate immunity and host energy homeostasis. Activated GPR43 suppresses the cAMP level and induces Ca2+. flux via coupling to G alpha, and G alpha(q), families, respectively. Additionally, GPR43 is reported to facilitate phosphorylation of ERK through G-protein-dependent pathways and interacts with 11-arrestin 2 to inhibit NF-kappa B signaling. However, other G-protein-dependent and independent signaling pathways involving GPR43 remain to be established. Here, we have demonstrated that GPR43 augments Rho GTPase signaling. Acetate and a synthetic agonist effectively activated RhoA and stabilized YAP/TAZ transcriptional coactivators through interactions of GPR43 with G alpha(q/11) and G alpha(12/13). Acetate-induced nuclear accumulation of YAP was blocked by a GPR43-specific inverse agonist. The target genes induced by YAP/TAZ were further regulated by GPR43. Moreover, in THP-1-derived M1-like macrophage cells, the Rho-YAP/TAZ pathway was activated by acetate and a synthetic agonist. Our collective findings suggest that GPR43 acts as a mediator of the Rho-YAP/TAZ pathway.

Type 2 diabetes mellitus may result from insulin resistance in skeletal muscle. Prokineticin receptor 1 (Prokr1) improves metabolic phenotype in adipose tissue and the cardiovascular system; however, its effects on skeletal muscle have not been investigated. We investigated the Prokr1 signaling pathways and its metabolic function in murine myoblast, satellite cells, and their differentiated myotubes. We measured the expression levels of Prokr1 in the skeletal muscle of mice as well as human skeletal muscle cell-derived myotubes. Prokineticin 2 (PROK2), a ligand of PROKR1, induced calcium mobilization in a dose-dependent manner and altered the mRNA levels of 578 genes in PROKR1-overexpressed HEK293T cells. Functional enrichment of differentially expressed genes revealed that PROKR1 activated Gq-mediated PI3K/AKT and MAPK/ERK signaling pathways in skeletal muscle cells. Prokr1 significantly activated the PI3K/AKT signaling pathway in myotubes derived from C2C12 and satellite cells, regardless of the presence or absence of insulin. Prokr1 also promoted the translocation of glucose transporter 4 (GLUT4) into the plasma membrane. In palmitate-induced insulin-resistant myotubes, Prokr1 enhanced insulin-stimulated AKT phosphorylation, GLUT4 translocation, and glucose uptake. mRNA and protein levels of Prokr1 were significantly decreased in skeletal muscle and white adipose tissue of diet-induced obese mice, and the amount of PROKR1 protein was significantly decreased in human skeletal muscle cell-derived myotubes under insulin resistance conditions. Taken together, these results demonstrate that Prokr1 plays an important role in insulin sensitivity and is a potential therapeutic target to ameliorate insulin resistance in skeletal muscle.

NF-κB signaling through both canonical and non-canonical pathways plays a central role in immune responses and inflammation. NF-κB-inducing kinase (NIK) stabilization is a key step in activation of the non-canonical pathway and its dysregulation implicated in various hematologic malignancies. The tumor suppressor, p53, is an established cellular gatekeeper of proliferation. Abnormalities of the TP53 gene have been detected in more than half of all human cancers. While the non-canonical NF-κB and p53 pathways have been explored for several decades, no studies to date have documented potential cross-talk between these two cancer-related mechanisms. Here, we demonstrate that p53 negatively regulates NIK in an miRNA-dependent manner. Overexpression of p53 decreased the levels of NIK, leading to inhibition of the non-canonical NF-κB pathway. Conversely, its knockdown led to increased levels of NIK, IKKα phosphorylation, and p100 processing. Additionally, miR-34b induced by nutlin-3 directly targeted the coding sequences (CDS) of NIK. Treatment with anti-miR-34b-5p augmented NIK levels and subsequent non-canonical NF-κB signaling. Our collective findings support a novel cross-talk mechanism between non-canonical NF-κB and p53.

Phosphorylation of the signaling component by protein kinase often leads to a kinase cascade or feedback loop. 3-Phosphoinositide-dependent kinase 1 (PDK1) signaling pathway diverges into various kinases including Akt and p70 S6 kinase (p70S6k). However, the PDK1 feedback mechanism remains elusive. Here, we demonstrated that UNC-51-like kinase (ULK1), an autophagy initiator kinase downstream of mechanistic target of rapamycin (mTOR), directly phosphorylated PDK1 on serine 389 at the linker region. Furthermore, our data showed that this phosphorylation affected the kinase activity of PDK1 toward downstream substrates. These results suggest a possible negative feedback loop between PDK1 and ULK1.

The mammalian Target of Rapamycin (mTOR) pathway regulates a variety of physiological processes, including cell growth and cancer progression. The regulatory mechanisms of these signals are extremely complex and comprise many feedback loops. Here, we identified the deubiquitinating enzyme ovarian tumor domain-containing protein 5 (OTUD5) as a novel positive regulator of the mTOR complex (mTORC) 1 and 2 signaling pathways. We demonstrated that OTUD5 stabilized β-transducin repeat-containing protein 1 (βTrCP1) proteins via its deubiquitinase (DUB) activity, leading to the degradation of Disheveled, Egl-10, and pleckstrin domain-containing mTOR-interacting protein (DEPTOR), which is an inhibitory protein of mTORC1 and 2. We also showed that mTOR directly phosphorylated OTUD5 and activated its DUB activity. RNA sequencing analysis revealed that OTUD5 regulates the downstream gene expression of mTOR. Additionally, OTUD5 depletion elicited several mTOR-related phenotypes such as decreased cell size and increased autophagy in mammalian cells as well as the suppression of a dRheb -induced curled wing phenotype by RNA interference of Duba , a fly ortholog of OTUD5 , in Drosophila melanogaster . Furthermore, OTUD5 knockdown inhibited the proliferation of the cancer cell lines with mutations activating mTOR pathway. Our results suggested a positive feedback loop between OTUD5 and mTOR signaling pathway.

Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), regulates genes involved in cell lineage and differentiation through methylating lysine 27 on histone H3 (H3K27me3). Recurrent gain-of-function mutations of EZH2 have been identified in various cancer types, in particular, diffuse large B-cell lymphoma (DLBCL), through large-scale genome-wide association studies and EZH2 depletion or pharmacological inhibition has been shown to exert an antiproliferative effect on cancer cells, both in vitro and in vivo. In the current study, a combination of pomalidomide and GSK126 synergistically inhibited the growth of EZH2 gain-of-function mutant Diffuse large B-cell lymphoma (DLBCL) cells. Furthermore, this synergistic effect appeared to be dependent on cereblon (CRBN), a cellular receptor of pomalidomide, but not degradation of IKAROS family zinc finger 1 (IKZF1) or IKAROS family zinc finger 3 (IKZF3). RNA sequencing analyses revealed that co-treatment with GSK126 and pomalidomide induced specific gene sets involved in B-cell differentiation and apoptosis. Synergistic growth inhibition and B-cell differentiation were further validated in xenograft mouse models. Our collective results provide a molecular basis for the mechanisms underlying the combined therapeutic effects of PRC2 inhibitors and pomalidomide on EZH2-mutated DLBCL.

Aryl hydrocarbon receptor nuclear translocator (ARNT) plays an essential role in maintaining cellular homeostasis in response to environmental stress. Under conditions of hypoxia or xenobiotic exposure, ARNT regulates the subset of genes involved in adaptive responses, by forming heterodimers with hypoxia-inducible transcription factors (HIF1α and HIF2α) or aryl hydrocarbon receptor (AhR). Here, we have shown that ARNT interacts with DDB1 and CUL4-associated factor 15 (DCAF15), and the aryl sulfonamides, indisulam and E7820, induce its proteasomal degradation through Cullin-RING finger ligase 4 containing DCAF15 (CRL4DCAF15) E3 ligase. Moreover, the two known neo-substrates of aryl sulfonamide, RNA-binding motif protein 39 (RBM39) and RNA-binding motif protein 23 (RBM23), are not required for ARNT degradation. In line with this finding, aryl sulfonamides inhibited the transcriptional activities of HIFs and AhR associated with ARNT. Our results collectively support novel regulatory roles of aryl sulfonamides in both hypoxic and xenobiotic responses.

Immunomodulatory drugs (IMiDs) exert anti-myeloma activity by binding to the protein cereblon (CRBN) and subsequently degrading IKZF1/3. Recently, their ability to recruit E3 ubiquitin ligase has been used in the proteolysis targeting chimera (PROTAC) technology. Herein, we design and synthesize a novel IMiD analog TD-106 that induces the degradation of IKZF1/3 and inhibits the proliferation of multiple myeloma cells in vitro as well as in vivo. Moreover, we demonstrate that TD-428, which comprises TD-106 linked to a BET inhibitor, JQ1 efficiently induce BET protein degradation in the prostate cancer cell line 22Rv1. Consequently, cell proliferation is inhibited due to suppressed C-MYC transcription. These results, therefore, firmly suggest that the newly synthesized IMiD analog, TD-106, is a novel CRBN modulator that can be used for targeted protein degradation.

Proteolysis targeting chimeras (PROTACs) are an emerging strategy for promoting targeted protein degradation by inducing the proximity between targeted proteins and E3 ubiquitin ligases. Although successful degradation of numerous proteins by PROTACs has been demonstrated, the elements that determine the degradability of PROTAC-targeted proteins have not yet been explored. In this study, we developed von Hippel-Lindau-Cereblon (VHL-CRBN) heterodimerizing PROTACs that induce the degradation of CRBN, but not VHL. A quantitative proteomic analysis further revealed that VHL-CRBN heterodimerizing PROTACs induced the degradation of CRBN, but not the well-known immunomodulatory drug (IMiD) neo-substrates, IKAROS family zinc finger 1 (IKZF1) and −3 (IZKF3). Moreover, truncation of disordered regions of CRBN and the androgen receptor (AR) attenuated their PROTAC-induced degradation, and attachment of the disordered region to stable CRBN or AR facilitated PROTAC-induced degradation. Thus, these results suggest that the intrinsically disordered region of targeted proteins is essential for efficient proteolysis, providing a novel criterion for choosing degradable protein targets.