c-Met Signaling Research Articles

Xanthohumol overcomes osimertinib resistance via governing ubiquitination-modulated Ets-1 turnover

Non-small cell lung cancer (NSCLC) is a prevalent and fatal malignancy with a significant global impact. Recent advancements have introduced targeted therapies like tyrosine kinase inhibitors (TKIs) such as osimertinib, which have improved patient outcomes, particularly in those with EGFR mutations. Despite these advancements, acquired resistance to TKIs remains a significant challenge. Hence, one of the current research priorities is understanding the resistance mechanisms and identifying new therapeutic targets to improve therapeutic efficacy. Herein, we identified high expression of c-Met in osimertinib-resistant NSCLC cells, and depletion of c-Met significantly inhibited the proliferation of osimertinib-resistant cells and prolonged survival in mice, suggesting c-Met as an attractive therapeutic target. To identify effective anti-tumor agents targeting c-Met, we screened a compound library containing 641 natural products and found that only xanthohumol exhibited potent inhibitory effects against osimertinib-resistant NSCLC cells. Moreover, combination treatment with xanthohumol and osimertinib sensitized osimertinib-resistant NSCLC cells to osimertinib both in vitro and in vivo. Mechanistically, xanthohumol disrupted the interaction between USP9X and Ets-1, and inhibited the phosphorylation of Ets-1 at Thr38, promoting its degradation, thereby targeting the Ets-1/c-Met signaling axis and inducing intrinsic apoptosis in osimertinib-resistant NSCLC cells. Overall, the research highlights the critical role of targeting c-Met to address osimertinib resistance in NSCLC. By demonstrating the efficacy of xanthohumol in overcoming resistance and enhancing therapeutic outcomes, this study provides valuable insights and potential new strategies for improving the clinical management of NSCLC.

Cigarette smoke extract induces malignant transformation and DNA damage via c-MET phosphorylation in human bronchial epithelial cells

Cigarette smoke, a complex mixture produced by tobacco combustion, contains a variety of carcinogens and can trigger DNA damage. Overactivation of c-MET, a receptor tyrosine kinase, may cause cancer and cellular DNA damage, but the underlying mechanisms are unknown. In this work, we investigated the mechanisms of cigarette smoke extract (CSE) induced malignant transformation and DNA damage in human bronchial epithelial cells (BEAS-2B). The results demonstrated that CSE treatment led to up-regulated mRNA expression of genes associated with the c-MET signaling pathway, increased expression of the DNA damage sensor protein γ-H2AX, and uncontrolled proliferation in BEAS-2B cells. ATR, ATR, and CHK2, which are involved in DNA damage repair, as well as the phosphorylation of c-MET and a group of kinases (ATM, ATR, CHK1, CHK2) involved in the DNA damage response were all activated by CSE. In addition, CSE activation promotes the phosphorylation modification of ATR, CHK1 proteins associated with DNA damage repair. The addition of PHA665752, a specific inhibitor of c-MET, or knock-down with c-MET both attenuated DNA damage, while overexpression of c-MET exacerbated DNA damage. Thus, c-MET phosphorylation may be involved in CSE-induced DNA damage, providing a potential target for intervention in the prevention and treatment of smoking-induced lung diseases.

Unveiling the Role of HGF/c-Met Signaling in Non-Small Cell Lung Cancer Tumor Microenvironment.

Non-small cell lung cancer (NSCLC) is characterized by several molecular alterations that contribute to its development and progression. These alterations include the epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), human epidermal growth factor receptor 2 (HER2), and mesenchymal-epithelial transition factor (c-MET). Among these, the hepatocyte growth factor (HGF)/c-MET signaling pathway plays a crucial role in NSCLC. In spite of this, the involvement of the HGF/c-MET signaling axis in remodeling the tumor microenvironment (TME) remains relatively unexplored. This review explores the biological functions of the HGF/c-MET signaling pathway in both normal and cancerous cells, examining its multifaceted roles in the NSCLC tumor microenvironment, including tumor cell proliferation, migration and invasion, angiogenesis, and immune evasion. Furthermore, we summarize the current progress and clinical applications of MET-targeted therapies in NSCLC and discuss future research directions, such as the development of novel MET inhibitors and the potential of combination immunotherapy.

Deregulated immune cell recruitment orchestrated by c-MET impairs pulmonary inflammation and fibrosis

BackgroundPulmonary fibrosis (PF) represents the pathologic end stage of several interstitial lung diseases (ILDs) associated with high morbidity and mortality rates. However, current treatments can only delay disease progression rather than provide a cure. The role of inflammation in PF progression is well-established, but new insights into immune regulation are fundamental for developing more efficient therapies. c-MET signaling has been implicated in the migratory capacity and effector functions of immune cells. Nevertheless, the role of this signaling pathway in the context of PF-associated lung diseases remains unexplored.MethodsTo determine the influence of c-MET in immune cells in the progression of pulmonary fibrosis, we used a conditional deletion of c-Met in immune cells. To induce pulmonary fibrosis mice were administered with bleomycin (BLM) intratracheally. Over the course of 21 days, mice were assessed for weight change, and after euthanasia at different timepoints, bronchoalveolar lavage fluid cells and lung tissue were assessed for inflammation and fibrosis. Furthermore, c-MET expression was assessed in cryobiopsy sections, bronchoalveolar lavage fluid cells samples and single cell RNA-sequencing dataset from human patients with distinct interstitial lung diseases.Resultsc-MET expression was induced in lung immune cells, specifically in T cells, interstitial macrophages, and neutrophils, during the inflammatory phase of BLM-induced PF mouse model. Deletion of c-Met in immune cells correlated with earlier weight recovery and improved survival of BLM-treated mice. Moreover, the deletion of c-Met in immune cells was associated with early recruitment of the immune cell populations, normally found to express c-MET, leading to a subsequent attenuation of the cytotoxic and proinflammatory environment. Consequently, the less extensive inflammatory response, possibly coupled with tissue repair, culminated in less exacerbated fibrotic lesions. Furthermore, c-MET expression was up-regulated in lung T cells from patients with fibrosing ILD, suggesting a potential involvement of c-MET in the development of fibrosing disease.ConclusionsThese results highlight the critical contribution of c-MET signaling in immune cells to their enhanced uncontrolled recruitment and activation toward a proinflammatory and profibrotic phenotype, leading to the exacerbation of lung injury and consequent development of fibrosis.

Selective inhibition of c-Met signaling pathways with a bispecific DNA nanoconnector for the targeted therapy of cancer

Hepatocyte growth factor receptor (c-Met) is a suitable molecular target for the targeted therapy of cancer. Novel c-Met-targeting drugs need to be developed because conventional small-molecule inhibitors and antibodies of c-Met have some limitations. To synthesize such drugs, we developed a bispecific DNA nanoconnector (STPA) to inhibit c-Met function. STPA was constructed by using DNA triangular prism as a scaffold and aptamers as binding molecules. After c-Met-specific SL1 and nucleolin-specific AS1411 aptamers were integrated with STPA, STPA could bind to c-Met and nucleolin on the cell membrane. This led to the formation of the c-Met/STPA/nucleolin complex, which in turn blocked c-Met activation. In vitro experiments showed that STPA could not only inhibit the c-Met signaling pathways but also facilitate c-Met degradation through lysosomes. STPA also inhibited c-Met-promoted cell migration, invasion, and proliferation. The results of in vivo experiments showed that STPA could specifically target to tumor site in xenograft mouse model, and inhibit tumor growth with low toxicity by downregulating c-Met pathways. This study provided a novel and simple strategy to develop c-Met-targeting drugs for the targeted therapy of cancer.

AHNAK2 Promotes the Progression of Pancreatic Ductal Adenocarcinoma by Maintaining the Stability of c-MET.

Pancreatic ductal adenocarcinoma (PDAC) is extremely malignant and rapidly progresses. The overall response rate of PDAC to current treatment methods is still unsatisfactory. Thus, identifying novel targets and clarifying the underlying mechanisms associated with PDAC progression may potentially offer additional treatment strategies. AHNAK2 is aberrantly expressed in a variety of tumors and exerts pro-tumorigenic effects. However, the biological role of AHNAK2 in PDAC remains poorly understood. The expression of AHNAK2 in PDAC and paired non-tumor tissues was detected by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). Lentivirus knockdown was performed to investigate the impact of AHNAK2 on the biological function of pancreatic cancer cells. The subcutaneous cell-derived xenograft (CDX) model and the KPC spontaneous mouse model with AHNAK2 silencing were used to observe the effects of AHNAK2 on tumor growth and prognosis. The expression of c-MET at protein level in response to HGF treatment was assessed using western blot. Our results demonstrated that AHNAK2 was highly expressed in PDAC clinical samples and associated with poor prognosis. Knockdown of AHNAK2 significantly inhibited the proliferation, migration, and invasion of pancreatic cancer cells. AHNAK2 knockdown or knockout resulted in tumor growth suppression and prolonged survival in mice with PDAC. In addition, AHNAK2 and c-MET expression levels showed a significant positive correlation at the post-transcriptional level. Mechanistically, AHNAK2 promoted tumor progression by preventing c-MET degradation and persistently activating the HGF/c-MET signaling pathway. Overall, our study revealed that AHNAK2 plays an important role in PDAC progression by modulating the c-MET signaling pathway, and targeting AHNAK2 may be an effective therapeutic strategy for PDAC.

Abstract LB025: CKD-703, a novel antibody-drug conjugate targeting cMET with an enhanced therapeutic index in solid tumors

Abstract In our pursuit of advancing cancer therapeutics, we introduce CKD-703, a groundbreaking cMET-targeting antibody drug conjugate (ADC) designed to strategically address the challenges posed by cMET overexpression and alteration in various solid tumors. Recognizing the pivotal role of tolerability and efficacy in shaping the therapeutic landscape of ADC, CKD-703 represents a significant advancement, aiming to widen the therapeutic index of cMET-targeted therapies. As we develop CKD-703, an innovative cMET-targeting ADC, we acknowledge the oncogenic potential of cMET alterations, particularly in non-small cell lung cancer (NSCLC), and the prevalent elevation of cMET across various cancers. Antibody drug conjugates targeting cMET (cMET-ADCs) have shown its therapeutic potential in treating cMET positive tumors, irrespective of their dependency on cMET signaling. Clinical success, however, has often been limited to NSCLC patients with the highest cMET levels. In light of this, CKD-703 is crafted to not only improve tolerability but also enhance efficacy, thereby broadening its application to a diverse patient population, including those with moderate cMET expression. To develop a novel cMET-ADC, Chong Kun Dang leverages Synaffix's ADC platform technology, incorporating glycan remodeling to achieve site-specific conjugation of the linker-payload to the antibody and ensuring a homogeneous drug-antibody ratio. These unique characteristics enhance CKD-703's stability in the bloodstream, minimizing the concentration of liberated free payload and significantly improving tolerability. The uniform DAR of CKD-703 contributes to improved pharmacokinetics, ensuring a consistent and sustained therapeutic effect. In comparison to benchmark antibodies, CKD-703 exhibits superior binding affinity to cMET and efficient internalization, resulting in enhanced therapeutic efficacy. This is evidenced in preclinical experiments which performed in several animal models that can mimic the complexities of cMET positive tumors with varying expression levels. The GLP toxicity studies of CKD-703 are ongoing. In conclusion, CKD-703 is a novel cMET-targeting ADC designed to overcome the limitations observed in existing cMET-ADCs in clinical trials as well as significantly improve tolerability and efficacy. By strategically addressing challenges associated with moderate cMET expression levels, CKD-703 emerges as a promising therapeutic candidate, aiming to broaden the therapeutic index in cMET-overexpressing solid tumors. This study highlights the importance of tailored approaches to diverse patient populations in advancing the development of CKD-703 in the pursuit of precision cancer therapies. Citation Format: Kwanwoo Lee, Sue-Youn Kim, Hye-Jin Hong, Eunjeong Park, Jungok Park, Seong-Ho Hong, Daekwon Bae, Nina Ha, Sohn-Ji Yeon, Inchang Hwang, Semi Kim, Yoonseok Lee, Ju-Hee Lee. CKD-703, a novel antibody-drug conjugate targeting cMET with an enhanced therapeutic index in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB025.

Abstract 1907: MYTX-011: A pH-dependent anti-cMET ADC with increased payload delivery in vivo and potent activity against cMET-expressing tumors of various epithelial origins

Abstract cMET is an oncogenic receptor tyrosine kinase overexpressed in multiple types of solid tumors, including non-small cell lung cancer (NSCLC), gastric, head and neck, liver cancer and others. Several cMET-directed therapies including antibody-drug conjugates (ADCs) have been developed that may benefit a subset of patients with tumors that have high cMET expression, MET amplification, or dependency on cMET signaling. MYTX-011 is an investigational, pH-sensitive, vcMMAE-based ADC that is designed to potentially benefit not only patients whose tumors express high levels of cMET, but also a broader set of patients whose tumors express low to moderate levels of cMET. MYTX-011 is engineered to rapidly dissociate from cMET only at the acidic pH of endolysosomes. We have previously demonstrated that MYTX-011 drives increased internalization and cytotoxicity in tumor cells expressing moderate cMET levels compared to a matched parent non-pH sensitive ADC. MYTX-011 also demonstrated markedly superior efficacy in NSCLC xenografts with moderate cMET expression as compared to either the parent ADC or benchmark ADCs representing clinical-stage anti-cMET ADCs with vcMMAE and maytansinoid payloads. MYTX-011 was efficacious in NSCLC PDX models with moderate cMET expression of adenocarcinoma/squamous histology and in heterogeneous cMET-expressing EGFR mutant models. In these studies, we demonstrate that MYTX-011 exerts enhanced efficacy by delivering increased levels of payload to cMET+ tumors compared to the parent ADC. MYTX-011 treatment induced significantly increased levels of phospho-histone H3 (pHH3), a pharmacodynamic marker of MMAE cytotoxicity, in moderate cMET expressing NCI-H1975 NSCLC xenograft tumors compared to the parent ADC. Further, increased levels of free MMAE were detected in tumors from mice treated with MYTX-011 compared to a matched dose of the parent ADC, indicating that the pH dependent binding engineered into MYTX-011 translated to increased payload delivery to tumors in vivo. To explore the potential efficacy of MYTX-011 in other cMET-expressing tumor types, we tested the activity of MYTX-011 in a panel of cell lines derived from gastric and head and neck cancers. MYTX-011 demonstrated increased cMET-dependent cytotoxicity compared to the parent ADC. MYTX-011 was also highly active in cMET+ xenograft models derived from gastric (non-MET amplified), esophageal, and head and neck cancers. Together, these findings highlight the potential of MYTX-011 as a therapeutic candidate for treating a broader range of cMET-expressing malignancies. Citation Format: Deepak Kanojia, William Comb, William Israelsen, Federico Colombo, Lin Nie, Nimish Gera, Thomas Chittenden, Brian Fiske. MYTX-011: A pH-dependent anti-cMET ADC with increased payload delivery in vivo and potent activity against cMET-expressing tumors of various epithelial origins [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 1907.

Abstract 5848: Unraveling c-MET targeting resistance mechanisms in gastric cancer via single-cell transcriptome analysis; Novel therapeutic target discovery of ERO1A and VEGFA

Abstract Background: Gastric cancer (GC) is the fifth most prevalent malignant tumor worldwide, and the fourth most common cause of mortality. c-MET has been reported to be associated with poor prognosis in GC. Over the decades, treatment approaches targeting HGF/c-MET have been developed including small molecule tyrosine kinase inhibitors, anti-c-MET and anti-HGF monoclonal antibodies. Unfortunately, the results of previous study support the development of acquired resistance to HGF/c-Met inhibitors. They suggest a possible resistance mechanism involving poor MET-status detection, crosstalk between c-MET and compensatory signaling pathways, the occurrence of acquired a new mutation, however, knowledge regarding the mechanism of c-MET inhibitor is limited.2. Purpose: We identify new markers and mechanisms that induce drug resistance when c-MET-targeted inhibitors are treated in c-MET-amplified GC cell lines in 3D culture, and discover new therapeutic targets for GC via single cell-RNA sequencing analysis.3. Material and Methods: Single cell-RNA sequencing is performed on c-MET amplified GC cell line in the 2D and 3D culture methods according to the presence or absence of c-MET inhibitor treatment. Through DEG and clustering analysis, the candidate genes estimated to impart resistance are selected and verified by qPCR. Through over-expression, induction of resistance for c-MET inhibitor treatment with target gene is evaluated in c-MET amplified GC cell line. c-MET inhibitor selected gene is constructed as a xenograft model to reveal the mechanism of drug resistance. Retrospective analysis confirms the relationship between target gene expression level and the resistance for c-MET inhibitor or other TKI treated patients with GC. 4. Results: Treatment of c-MET-amplified GC cell lines with c-MET inhibitors under the conditions of 2D and 3D cultures confirmed that resistance was induced only in the 3D. We performed a high-precision single cell RNA-Seq analysis of 18056 single cells in total from c-MET amplified GC cell line MKN-45 in 2D/3D culture control as well as corresponding samples with volitinib treatment investigate their gene expression signatures. Fifteen selected candidate genes presumed to induce drug resistance were screened and validated by qPCR, with a total of six genes consistent with the sequencing results. After over-expression of six genes to the c-MET amplified gastric cancer cell line, c-MET inhibitors were treated and cell viability was performed to check for resistance to cell viability. Finally, ERO1A and VEGFA were over-expressed to confirm the resistant for the c-MET inhibitor treatment in vitro and in vivo.5. Discussion: We plan to build a xenograft mouse model with candidate genes such as ERO1a and VEGFA to prove whether the candidate gene overcomes drug resistance when they are on or off. Citation Format: Hajung Kim, Jinhyeon Kim, Seung-Jae Myung, Charny Park, Sun young Kim. Unraveling c-MET targeting resistance mechanisms in gastric cancer via single-cell transcriptome analysis; Novel therapeutic target discovery of ERO1A and VEGFA [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 5848.

Utilizing bivalent aptamers as first DNA agonist to activate RTKs heterodimer of different families

Heterodimerization in RTKs is of vital importance in the RTK signaling and cell functions. Heterodimerization between RTKs can result in diversity of downstream signals, increasing the ability of cells to respond to external experiments. Traditional RTKs heterodimerization always occur in the same families and is lack of agonists to activate the heterodimeric RTKs signaling pathway. Herein, we developed the DNA agonist based on bivalent aptamers for the heterodimerized RTKs of different families, AF/AM-1, which could simultaneously activate FGFR1 and c-Met signaling. It is the first agonist that realizing the heterodimerization and activation of FGFR1 and c-Met, two different RTK families. The activation of FGFR1/c-Met heterodimer result in the down-stream signals transduction, such as the phosphorylation of Akt and Erk, inducing the cell migration and proliferation. The DNA agonist for RTK heterodimer of different families would have potential applications in the fields of biomedicine.

Loss of LECT2 promotes ovarian cancer progression by inducing cancer invasiveness and facilitating an immunosuppressive environment

Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine that can bind to several receptors and mediate distinct molecular pathways in various cell settings. Changing levels of LECT2 have been implicated in multiple human disease states, including cancers. Here, we have demonstrated reduced serum levels of LECT2 in patients with epithelial ovarian cancer (EOC) and down-regulated circulating Lect2 as the disease progresses in a syngeneic mouse ID8 EOC model. Using the murine EOC model, we discovered that loss of Lect2 promotes EOC progression by modulating both tumor cells and the tumor microenvironment. Lect2 inhibited EOC cells’ invasive phenotype and suppressed EOC’s transcoelomic metastasis by targeting c-Met signaling. In addition, Lect2 downregulation induced the accumulation and activation of myeloid-derived suppressor cells (MDSCs). This fostered an immunosuppressive microenvironment in EOC by inhibiting T-cell activation and skewing macrophages toward an M2 phenotype. The therapeutic efficacy of programmed cell death-1 (PD-1)/PD-L1 pathway blockade for the ID8 model was significantly hindered. Overall, our data highlight multiple functions of Lect2 during EOC progression and reveal a rationale for synergistic immunotherapeutic strategies by targeting Lect2.

Hovenia dulcis Suppresses the Growth of Huh7-Derived Liver Cancer Stem Cells by Inducing Necroptosis and Apoptosis and Blocking c-MET Signaling.

Liver cancer stem cells (LCSCs) contribute to the initiation, metastasis, treatment resistance, and recurrence of hepatocellular carcinoma (HCC). Therefore, exploring potential anticancer agents targeting LCSCs may offer new therapeutic options to overcome HCC treatment failure. Hovenia dulcis Thunberg (HDT), a tree from the buckthorn family found in Asia, exhibits various biological activities, including antifatigue, antidiabetic, neuroprotective, hepatoprotective, and antitumor activities. However, the therapeutic effect of HDT in eliminating LCSCs remains to be confirmed. In this study, we evaluated the inhibitory activity of ethanol, chloroform, and ethyl acetate extracts from HDT branches on the growth of Huh7-derived LCSCs. The ethyl acetate extract of HDT (EAHDT) exhibited the most potent inhibitory activity against the growth of Huh7 LCSCs among the three HDT extracts. EAHDT suppressed the in vitro self-renewal ability of Huh7 LCSCs and reduced tumor growth in vivo using the Huh7 LCSC-transplanted chick embryo chorioallantoic membrane model. Furthermore, EAHDT not only arrested the cell cycle in the G0/G1 phase but also induced receptor-interacting protein kinase 3/mixed-lineage kinase domain-like protein-mediated necroptosis and caspase-dependent apoptosis in Huh7 LCSCs in a concentration-dependent manner. Furthermore, the growth inhibitory effect of EAHDT on Huh7 LCSCs was associated with the downregulation of c-MET-mediated downstream signaling pathways and key cancer stemness markers. Based on these findings, we propose that EAHDT can be used as a new natural drug candidate to prevent and treat HCC by eradicating LCSCs.

Oleocanthal, an Antioxidant Phenolic Compound in Extra Virgin Olive Oil (EVOO): A Comprehensive Systematic Review of Its Potential in Inflammation and Cancer.

The Mediterranean diet is linked to various health benefits, especially the consumption of olive oil as a key component. Multiple studies highlight its advantages, particularly due to its fatty acid composition and additional components like phenolic compounds. A significant antioxidant compound, oleocanthal, known for its antioxidant properties, has gained attention in the pharmaceutical industry for its anti-inflammatory and antiproliferative effects. It shows promise in addressing cardiovascular diseases, metabolic syndrome, and neuroprotection. This systematic review aims to evaluate the existing literature on oleocanthal, examining its role in biological processes and potential impact on conditions like inflammation and cancer. We performed several searches in PubMed (MEDLINE), Web of Science (WOS), and Cochrane based on the terms "Oleocanthal", "Cancer", and "Inflammation". The inclusion criteria were as follows: studies whose main topics were oleocanthal and cancer or inflammation. On the other hand, the exclusion criteria were studies that were not focused on oleocanthal, reviews, or editorial material. Given that these findings are explanatory rather than derived from clinical trials, we refrained from employing methods to assess potential bias. This systematic review did not receive any external funding. We found 174 records from these searches, where we discarded reviews and editorial material, duplicated articles, and 1 retracted article. Finally, we had 53 reports assessed for eligibility that were included in this review. OC exhibits promising therapeutic potential against both inflammation and cancer. We addressed its ability to target inflammatory genes and pathways, offering potential treatments for conditions like rheumatic diseases by regulating pathways such as NF-kB and MAPK. Additionally, OC's anticancer properties, particularly its notable inhibition of c-Met signaling across various cancers, highlight its efficacy, showcasing promise as a potential treatment.

Cell Membrane-Anchored DNA Nanoinhibitor for Inhibition of Receptor Tyrosine Kinase Signaling Pathways via Steric Hindrance and Lysosome-Induced Protein Degradation.

Receptor tyrosine kinase (RTK) plays a crucial role in cancer progression, and it has been identified as a key drug target for cancer targeted therapy. Although traditional RTK-targeting drugs are effective, there are some limitations that potentially hinder the further development of RTK-targeting drugs. Therefore, it is urgently needed to develop novel, simple, and general RTK-targeting inhibitors with a new mechanism of action for cancer targeted therapy. Here, a cell membrane-anchored RTK-targeting DNA nanoinhibitor is developed to inhibit RTK function. By using a DNA tetrahedron as a framework, RTK-specific aptamers as the recognition elements, and cholesterol as anchoring molecules, this DNA nanoinhibitor could rapidly anchor on the cell membrane and specifically bind to RTK. Compared with traditional RTK-targeting inhibitors, this DNA nanoinhibitor does not need to bind at a limited domain on RTK, which increases the possibilities of developing RTK inhibitors. With the cellular-mesenchymal to epithelial transition factor (c-Met) as a target RTK, the DNA nanoinhibitor can not only induce steric hindrance effects to inhibit c-Met activation but also reduce the c-Met level via lysosome-mediated protein degradation and thus inhibition of c-Met signaling pathways and related cell behaviors. Moreover, the DNA nanoinhibitor is feasible for other RTKs by just replacing aptamers. This work may provide a novel, simple, and general RTK-targeting nanoinhibitor and possess great value in RTK-targeted cancer therapy.

Manipulating HGF signaling reshapes the cirrhotic liver niche and fills a therapeutic gap in regeneration mediated by transplanted stem cells

Long-term stem cell survival in the cirrhotic liver niche to maintain therapeutic efficacy has not been achieved. In a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis animal model, we previously showed that liver-resident stem/progenitor cells (MLpvNG2+ cells) or immune cells have improved survival in the fibrotic liver environment but died via apoptosis in the cirrhotic liver environment, and increased levels of hepatocyte growth factor (HGF) mediated this cell death. We tested the hypothesis that inhibiting HGF signaling during the cirrhotic phase could keep the cells alive. We used adeno-associated virus (AAV) vectors designed to silence the c-Met (HGF-only receptor) gene or a neutralizing antibody (anti-cMet-Ab) to block the c-Met protein in the DEN-induced liver cirrhosis mouse model transplanted with MLpvNG2+ cells between weeks 6 and 7 after DEN administration, which is the junction of liver fibrosis and cirrhosis at the site where most intrahepatic stem cells move toward apoptosis. After 4 weeks of treatment, the transplanted MLpvNG2+ cells survived better in c-Met-deficient mice than in wild-type mice, and cell activity was similar to that of the mice that received MLpvNG2+ cells at 5 weeks after DEN administration (liver fibrosis phase when most of these cells proliferated). Mechanistically, a lack of c-Met signaling remodeled the cirrhotic environment, which favored transplanted MLpvNG2+ cell expansion to differentiation into mature hepatocytes and initiate endogenous regeneration by promoting mature host hepatocyte generation and mediating functional improvements. Therapeutically, c-Met-mediated regeneration can be mimicked by anti-cMet-Ab to interfere functions, which is a potential drug for cell-based treatment of liver fibrosis/cirrhosis.

Abstract B124: MYTX-011 is a highly internalized ADC with anti-tumor activity across a spectrum of NSCLC preclinical models with various levels of cMET expression

Abstract cMET overexpression occurs in non-small cell lung cancer (NSCLC) and antibody-drug conjugates targeting cMET (cMET-ADCs) have been developed to treat cMET+ NSCLC tumors irrespective of dependency on cMET signaling. cMET-ADCs have shown promising clinical activity, but largely in a subset of NSCLC patients whose tumors express the highest cMET levels. MYTX-011, an investigational, pH-sensitive ADC incorporating the clinically validated vcMMAE linker-payload, was specifically designed to potentially benefit a broader population of patients including those whose tumors express lower cMET levels. We’ve previously demonstrated that pH-sensitive cMET binding of MYTX-011 drives higher internalization in cMET+ tumor cells, and greater cytotoxicity against a panel of cMET+ cancer cell lines in vitro. MYTX-011 also shows superior efficacy in NSCLC xenograft models with high or moderate cMET expression levels when compared to the matched non-pH-sensitive Parent ADC or a Benchmark ADC generated by hinge conjugation of a clinical stage anti-cMET ADC antibody to vcMMAE. Here we demonstrate that the MYTX-011 monoclonal antibody, which is engineered to selectively and rapidly lose affinity at the acidic pH of endolysosomes, rapidly internalizes and accumulates in cells expressing moderate cMET levels compared to the matched parent antibody. MYTX-011 in vitro cytotoxicity correlated with cMET expression levels and conferred sensitivity to cells that were otherwise insensitive to ADCs generated with the parent or Benchmark antibodies. Importantly, cells that were only sensitive to MYTX-011, but not matched controls, expressed low and moderate levels of cMET. Furthermore, MYTX-011 was active against the low cMET-expressing NCI-H2122 xenograft model in vivo whereas the Benchmark ADC was inactive. In the moderate cMET-expressing NCI-H1975 xenograft model, MYTX-011 was more active than both the Benchmark ADC and a second benchmark ADC generated by hinge conjugation of a clinical stage biparatopic anti-cMET ADC antibody to a maytansinoid linker/toxin. MYTX-011 activity was further evaluated against a panel of NSCLC patient derived xenograft (PDX) models with relevant histology, cMET expression, and genetic profiles. MYTX-011 was highly active in cMET+ squamous and adenocarcinoma PDX, EGFR WT and mutant PDX, in a PDX model with MET ex14 skipping mutation, and in PDX with heterogenous cMET expression. Together, these findings highlight the potential of MYTX-011 as a therapeutic candidate for treating a broader range of cMET+ NSCLC malignancies than other cMET-ADCs. Citation Format: William C Comb, Deepak Kanojia, Federico Colombo, Vijay Ramesh, Nimish Gera, Thomas Chittenden, Brian Fiske. MYTX-011 is a highly internalized ADC with anti-tumor activity across a spectrum of NSCLC preclinical models with various levels of cMET expression [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B124.

PIM1 Point Mutations Increase Migration of Diffuse Large B-Cell Lymphoma (DLBCL) Cells

PIM1 serine-threonine kinase is known proto-oncogene, highly expressed in lymphoid malignancies. It regulates crucial processes for the cancer cell, like proliferation, apoptosis, metabolism or migration. In DLBCLs, PIM1 gene is a target of somatic hypermutation and one of the most frequently mutated genes in DLBCL patients. PIM1 mutations are more frequent in ABC-type tumors, which have worse survival prognosis than GCB-type, and often co-occur with mutations in MYD88 and CD79B. Analyses of the available clinical data show that the most significant differences between the groups of DLBCL patients with or without PIM1 mutations are tumor mutational burden, age at diagnosis and extranodal (testicular) involvement. In line with this finding, PIM1, MYD88 and CD79B mutations have also been reported to be highly prevalent in extranodal B cell lymphomas, localized in central nervous system (PCNSL), testicles (PTL) or intravascular space (IVLBCL). Our goal was to assess the role of the most common PIM1 point mutations in the biology and pathogenesis of DLBCL cells. The HBL-1 cell line was chosen as a model of MYD88 L265P-positive ABC-DLBCL subtype. PIM1 knock-out was performed using CRISPR/Cas9 method. Sequences of wild type PIM1, kinase-dead PIM1K67M, serving as a an inactive kinase control, and 7 most frequent mutants (PIM1G28D, PIM1E70K, PIM1P81L, PIM1S97N, PIM1P125A, PIM1L164F, PIM1L184F) were reintroduced to the HBL-1 PIM1 KO cells using lentiviral vectors. PIM1 mutations did not affect the proliferation of DLBCL cells and did not increase resistance to doxorubicin, excluding the proliferation rate or treatment resistance as a cause of the poor prognosis associated with PIM1 mutations. We performed RNA-seq analysis of the HBL-1-PIM1-WT and HBL-1-PIM1-mutants. The most upregulated pathways in PIM1 mutants (including PIM1K67M) were those related to Interferon gamma, IL-15, STAT3, cMET and semaphorin-plexin signaling (involved in proliferation, actin dynamics and migration of cancer cells). We also found that PIM1 mutants universally overexpress cMET oncogene. Moreover, gene set and pathway enrichment analyses indicated that the PIM1-mutant cells exhibit features associated with altered actin polymerization, RHO GTPases and cell membrane dynamics, suggesting the increased migratory potential of the cells and their propensity to spread to extranodal sites. Higher surface level of cMET in PIM1 mutant cells was confirmed using FACS analysis. Since cMET and CXCR4 cooperate in signaling and trigger AKT/mTOR, which is required for CXCR4-mediated cell motility, we evaluated whether PIM1 mutant cells would exhibit increased phosphorylation of AKT. In line with this hypothesis, we noted higher pAKT and pERK1/2 in PIM1 mutant cells than in PIM1 WT cells. Finally, in transwell assays, PIM1 mutant cells exhibited markedly increased migratory potential. Taken together, these studies indicate that PIM1 mutants are not likely associated with differential drug responses, but rather facilitate tumor dissemination and are thus associated with inferior prognosis in DLBCL patients harboring PIM1 mutations.

PTPN23 ubiquitination by WDR4 suppresses EGFR and c-MET degradation to define a lung cancer therapeutic target

Aberrant overexpression or activation of EGFR drives the development of non-small cell lung cancer (NSCLC) and acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) by secondary EGFR mutations or c-MET amplification/activation remains as a major hurdle for NSCLC treatment. We previously identified WDR4 as a substrate adaptor of Cullin 4 ubiquitin ligase and an association of WDR4 high expression with poor prognosis of lung cancer. Here, using an unbiased ubiquitylome analysis, we uncover PTPN23, a component of the ESCRT complex, as a substrate of WDR4-based ubiquitin ligase. WDR4-mediated PTPN23 ubiquitination leads to its proteasomal degradation, thereby suppressing lysosome trafficking and degradation of wild type EGFR, EGFR mutant, and c-MET. Through this mechanism, WDR4 sustains EGFR and c-MET signaling to promote NSCLC proliferation, migration, invasion, stemness, and metastasis. Clinically, PTPN23 is downregulated in lung cancer and its low expression correlates with WDR4 high expression and poor prognosis. Targeting WDR4-mediated PTPN23 ubiquitination by a peptide that competes with PTPN23 for binding WDR4 promotes EGFR and c-MET degradation to block the growth and progression of EGFR TKI-resistant NSCLC. These findings identify a central role of WDR4/PTPN23 axis in EGFR and c-MET trafficking and a potential therapeutic target for treating EGFR TKI-resistant NSCLC.

Impact of SMAD2 and MET Expression on Lymph Node Metastasis of HER2-positive Gastric Cancer Cells.

Intra-tumoral heterogeneity, which is frequently found in various types of cancers, has been suggested to play an important role in cancer progression and metastasis. The findings of our previous study suggested that p-SMAD2 and c-MET signaling might play important roles in the progression to lymph node metastasis of HER2-positive gastric cancer. In this study, we confirmed the effect of SMAD2/MET signaling in the progression of HER2-positive gastric cancer in an animal model. NCI-N87 cells over-expressing ERBB2, SMAD2, MET were used. To confirm the role of SMAD2 and MET expression on lymph node metastasis of gastric cancer, we orthotopically injected NCI-N87 cells with or without the knockdown of both SMAD2 and MET into the gastric walls of BALBc nude mice. The number of metastatic lymph nodes was significantly smaller in the knockdown group compared to that in the control group. However, there was no significant difference in gastric tumor size between the two groups. SMAD2 and MET signaling might play important roles specifically in the progression to lymph node metastasis of HER2-positive gastric cancer. c-MET and SMAD2 may be useful targets for preventing lymph node metastasis in patients with HER2-positive gastric cancer.

A novel bivalent anti-c-MET/PD-1 bispecific antibody exhibits potent cytotoxicity against c-MET/PD-L1-positive colorectal cancer.

Previously, we generated a novel bispecific antibody (BsAb) simultaneously targeting both c-MET and PD-1 (PDCD1), which can bridge T cells and c-MET positive tumor cells. However, the specific mechanisms and antitumor activities of the BsAb against c-MET/PD-L1 (CD274) positive colorectal cancer (CRC) is not completely understood. In this study, in addition to the tumor intrinsic mechanism investigation with molecular biology assay in vitro, a humanized mouse model was used to evaluate antitumor activity of the BsAb in vivo. The BsAb could inhibit c-MET/PD-L1+ CRC cell migration and show strong antitumor activity against HCT116 tumors in mice, potentially by inducing the degradation of c-MET protein in a dose and time-dependent manner. The BsAb could suppress the phosphorylation of c-MET downstream proteins GRB2-associated-binding protein 1 (Gab1) and focal adhesion kinase (FAK). Considering the tumor extrinsic mechanism, the BsAb may promote phagocytosis of macrophage. Furthermore, the level of plasma exosomal-c-MET/PD-L1 is able to distinguish CRC patients from healthy controls. In summary, the BsAb exhibited potent anti-tumor activities by two distinguished mechanisms: inhibition of c-MET signal transduction and promotion of macrophage-mediated phagocytosis. Our BsAb may provide a novel therapeutic agent for patients with c-MET/PD-L1+ CRC, and the status of exosomal-c-MET/PD-L1 can serve as a biomarker to predict responsiveness to treatment of our BsAb.