NCI-N87 Cells Research Articles

Metformin in combination with chemotherapy increases apoptosis in gastric cancer cells and counteracts senescence induced by chemotherapy.

Gastric cancer (GC) is the fourth leading cause of cancer death in the world, and there is a demand for new therapeutic agents to treat GC. Metformin has been demonstrated to be an antineoplastic agent in some types of cancer; however, it has not been sufficiently valued in treating GC because the effect of metformin in combination with chemotherapy regimens has not yet been evaluated. The present study aimed to evaluate the mechanisms underlying cell death induced by metformin alone or when combined with chemotherapy. The cytogenetic characteristics of the NCI-N87 cell line were determined by fluorescence in situ hybridization (FISH). To determine viability, the cells were treated with metformin, epirubicin, cisplatin, docetaxel and 5-fluorouracil (individually and at different concentrations). Subsequently, the cells were treated with metformin alone, and in combination with the chemotherapeutic drugs and the epirubicin + cisplatin + 5-fluorouracil, docetaxel + cisplatin + 5-fluorouracil, and cisplatin + 5-fluorouracil regimens. Cell viability, proliferation and mitochondrial membrane potential (ΔΨm) were analyzed by spectrophotometry. Apoptosis, caspase activity and cell cycle progression were assessed by flow cytometry. Finally, light microscopy was used to evaluate senescence and clonogenicity. The results revealed that metformin, alone and when combined with chemotherapy, increased the proportion of apoptotic cells, promoted the loss of ΔΨm, and induced apoptosis through caspase activity in GC cells. Moreover, metformin decreased cell proliferation. In addition, metformin alone did not induce senescence and it counteracted the effects of chemotherapy-induced senescence in GC cells. Additionally, metformin, alone and when combined with chemotherapy, decreased the clonogenic capacity of NCI-N87 GC cells. In conclusion, metformin may increase the effects of chemotherapy on NCI-N87 cell death and could represent an option to improve the treatment of GC.

A Probe-Free Occupancy Assay to Assess a Targeted Covalent Inhibitor of Receptor Tyrosine-Protein Kinase erbB-2.

Establishing target engagement is fundamental to effective target-based drug development. It paves the way for efficient medicinal chemistry design and definitive answers about target validation in the clinic. For irreversible targeted covalent inhibitor (TCI) drugs, there is a unique opportunity to establish and quantify the target engagement or occupancy. This is typically accomplished by using a covalent molecular probe, often a TCI analogue, derivatized to allow unoccupied target sites to be tracked; the difference of total sites minus unoccupied sites yields the occupied sites. When such probes are not available or the target is not readily accessible to covalent probes, another approach is needed. Receptor tyrosine-protein kinase erbB-2 (HER2) occupancy by afatinib presents such a case. Available HER2 covalent probes were unable to consistently modify HER2 after sample preparation, resulting in inadequate data. We demonstrate an alternative quantitative probe-free occupancy (PFO) method. It employs the immunoprecipitation of HER2 and direct mass spectrometer analysis of the cysteine-containing peptide that is targeted and covalently occupied by afatinib. Nontarget HER2 peptides provide normalization to the total protein. We show that HER2 occupancy by afatinib correlates directly to the inhibition of the receptor tyrosine kinase activity in NCI-N87 cells in culture and in vivo using those cells in a mouse tumor xenograft mode.

RHBDD1 Promotes the Growth and Stemness Characteristics of Gastric Cancer Cells by Activating Wnt/β-catenin Signaling Pathway.

Gastric cancer (GC) is threatening public health, with at least one million new cases reported each year. Rhomboid domain-containing protein 1 (RHBDD1) has been identified to regulate the proliferation, migration, and metastasis of cancer cells. However, the role of RHBDD1 in GC has not been elucidated. This study aimed to investigate the role of RHBDD1 on the growth, metastasis, and stemness characteristics of GC. RHBDD1 expression was analyzed from the TCGA databank. qRT-PCR was conducted to evaluate the transcription level of RHBDD1. Western blots were used to evaluate the protein expression of RHBDD1, CD133, CD44, Nanog, β-catenin and c-myc. Colony formation assay and transwell assay were conducted to evaluate the growth and metastasis of NCI-N87 cells, respectively. Sphere-forming assay was performed to study the stemness characteristics. The nude mice xenotransplantation model and immunohistochemistry (IHC) were performed to evaluate the growth of GC in vivo. Results: RHBDD1 expression is elevated in GC cells and clinical tissues. RHBDD1 expression is positively associated with cell proliferation and metastasis of GC cells. RHBDD1 knockdown suppresses the expression of CD133, CD44 and Nanog and attenuates sphere-forming ability. RHBDD1 activates the Wnt/β-catenin pathway via promoting the expression of β-catenin / c-myc and inducing β-catenin translocation into nuclear. RHBDD1 knockdown inhibits the growth of GC in nude mice xenotransplantation model. RHBDD1 is highly expressed in GC, and its knockdown inhibits the growth, metastasis and stemness characteristics of GC cells through activating the Wnt/β-catenin pathway, suggesting that RHBDD1 has the potential to be a novel therapeutic target for GC treatment.

PD-L1 has a heterogeneous and dynamic expression in gastric cancer with implications for immunoPET.

This study aimed to investigate the dynamics of programmed death-ligand 1 (PD-L1) expression, spatial heterogeneity, and binding affinity of FDA-approved anti-PD-L1 antibodies (avelumab and atezolizumab) in gastric cancer. Additionally, we determined how PD-L1 glycosylation impacts antibody accumulation in gastric cancer cells. Dynamic PD-L1 expression was examined in NCIN87 gastric cancer cells. Comparative binding studies of avelumab and atezolizumab were conducted in gastric cancer models, both in vitro and in vivo. Antibody uptake in tumors was visualized through positron emission tomography (PET) imaging. PD-L1 glycosylation status was determined via Western blot analyses before and after PNGase F treatment. Consistent findings revealed time-dependent PD-L1 induction in NCIN87 gastric cancer cells and spatial heterogeneity in tumors, as shown by PET imaging and immunofluorescence. Avelumab displayed superior binding affinity to NCIN87 cells compared to atezolizumab, confirmed by in vivo PET imaging and ex vivo biodistribution analyses. Notably, PD-L1 glycosylation at approximately 50 kDa was observed, with PNGase F treatment inducing a shift to 35 kDa in molecular weight. Tissue samples from patient-derived xenografts (PDXs) validated the presence of both glycosylated and deglycosylated PD-L1 (degPD-L1) forms in gastric cancer. Immunofluorescence microscopy and binding assays demonstrated enhanced avelumab binding post-deglycosylation. This study provides an understanding of dynamic and spatially heterogeneous PD-L1 expression in gastric cancer. Anti-PD-L1 immunoPET was able to visualize gastric tumors, and PD-L1 glycosylation has significant implications for antibody recognition. These insights contribute to demonstrating the complexities of PD-L1 in gastric cancer, holding relevance for refining PD-L1 imaging-based approaches.

Ketohexokinase-A deficiency attenuates the proliferation via reducing β-catenin in gastric cancer cells

Overconsumption of fructose is closely related to cancer. Ketohexokinase (KHK) catalyzes the conversion from fructose to fructose-1-phosphate (F1P), which is the first and committed step of fructose metabolism. Recently, aberrant KHK activation has been identified in multiple malignancies. However, the roles of KHK in gastric cancer (GC) cells are largely unclear. Herein, we reveal that the expression of ketohexokinase-A (KHK-A), one alternatively spliced KHK isoform that possesses low affinity for fructose, was markedly increased in GC cells. Depletion of endogenous KHK-A expression using lentiviruses encoding short hairpin RNAs (shRNAs) or pharmaceutical disruption of KHK-A activity using KHK-IN-1 hydrochloride in GC NCI–N87 and HGC-27 cells inhibited the proliferation in vitro and in vivo. Additionally, the mitochondrial respiration in the GC cells with KHK-A deficiency compared with the control cells was significantly impaired. One commercially-available antibody array was used to explore the effects of KHK-A knockdown on signaling pathways, showing that β-catenin was remarkably reduced in the KHK-A deficient GC cells compared with the control ones. Pharmaceutical reduction in β-catenin levels slowed down the proliferation of GC cells. These data uncover that KHK-A promotes the proliferation in GC cells, indicating that this enzyme might be a promising therapeutical target for GC treatment.

Discovery of novel HER2 targeting peptide-camptothecin conjugates with effective suppression for selective cancer treatment

Due to the strong selectivity and permeability of tumor tissue, anti-cancer peptide-drug conjugates (PDCs) can accumulate high concentration of toxic payloads at the target, effectively killing tumor cells. This approach holds great promise for tumor-targeted treatment. In our previous study, we identified the optimal peptide P1 (NPNWGRSWYNQRFK) targeting HER2 from pertuzumab, a monoclonal antibody that blocks the HER2 signaling pathway. Here, a series of PDCs were constructed through connecting P1 and CPT with different linkers. Among these, Z8 emerged as the optimal compound, demonstrating good antitumor activity and targeting ability in biological activity tests. Z8 exhibited IC50 values of 1.04 ± 0.24 μM and 1.91 ± 0.71 μM against HER2-positive SK-BR-3 and NCI-N87 cells, respectively. Moreover, superior antitumor activity and higher biosafety of Z8 were observed compared to the positive control CPT in vivo, suggesting a novel idea for the construction of PDCs.

Investigating the individual and mixture cytotoxicity of co-occurring aflatoxin B1, enniatin B, and sterigmatocystin on gastric, intestinal, hepatic, and renal cellular models

This study investigates the individual and combined effects of the mycotoxins, Aflatoxin B1 (AFB1), Enniatin B (ENNB) and Sterigmatocystin (STG), on the cellular viability of gastric (NCI–N87), intestinal (Caco-2), hepatic (Hep-G2) and renal (Hek-293) cells, shedding light on synergistic or antagonistic effects using a constant ratio combination design proposed by Chou-Talalay. These toxins are prevalent in cereal-based foods, frequently consumed by children which raises concerns about their exposure to these mycotoxins. This population is particularly vulnerable to the effects of these toxins due to their underdeveloped organs and incompletely structured physiological processes. Results showed that ENB was the most toxic of the three mycotoxins across all cell lines, while STG and AFB1 showed lower toxicity. The combination of ENNB + STG was found to be the most potent in terms of binary mixtures. In regard to ternary combinations, Caco-2 cells are more sensitive to the tested mycotoxins, whereas NCI–N87 cells show lower levels of cell damage. Worrying dose reduction values (>10-fold) were found for ENNB in binary and ternary combinations at low exposure levels. These findings are significant for establishing initial reference values, which play a pivotal role in estimating reference doses that are subsequently incorporated into the broader risk assessment process.

Abstract 5232: Placental circulating T cells expressing CD16 in combination with trastuzumab demonstrate robust anti-tumor antibody-dependent cellular cytotoxicity (ADCC) against gastric cancer

Abstract Introduction: Celularity is developing PT-CD16VS as a novel platform for use in combination with approved monoclonal antibodies for the potential treatment of multiple cancers. PT-CD16VS is an allogeneic cell therapy derived from human postpartum placental circulating T (P-T) cells that are genetically modified to express a proprietary CD16 variant and endogenous T cell receptor (TCR) knockout. Here we report the characterization and preclinical evaluation of PT-CD16VS against HER2+ gastric cancer cells in combination with Trastuzumab. In vitro safety studies evaluating the potential for on-target/off-tumor activity of PT-CD16VS against HER2 low expressing lung epithelial cells (LEC) and human dermal fibroblasts (HDF) are also highlighted. Methods: P-T cells were activated and transduced with a lentiviral vector containing a CD16 construct expressing a high affinity CD16 variant, CD16VS. Following transduction, P-T cells were transfected to knock out the TCR. In vitro, the functional activity of PT-CD16VS cells in combination with Trastuzumab was assessed against the HER2+ gastric cancer NCI-N87 cell line in cytotoxicity, cytokine release, and proliferation assays. For on-target/off-tumor studies, similar assays were utilized to assess activity against the HER2 low expressing LEC and HDF cell lines. In vivo, PT-CD16VS with Trastuzumab was evaluated in a subcutaneous NCI-N87 xenograft model in NSG mice. Tumor volume was measured and tumor samples were evaluated for PT-CD16VS infiltration. Results: High CD16 transduction efficiency (>60%) was achieved across donors. In vitro, PT-CD16VS plus Trastuzumab demonstrated potent cytotoxicity against NCI-N87 cells at low E:T ratios (0.625:1) and at 12 hours of co-culture with moderate cytokine secretion at 24-hour of co-culture. PT-CD16VS proliferated following 48-hour incubation with NCI-N87 cells and Trastuzumab, as compared to an IgG1 control. PT-CD16VS in combination with Trastuzumab did not elicit on-target/off-tumor cytotoxicity or cytokine release in response to low HER2 expression on normal LEC or HDF cells. In vivo, PT-CD16VS with Trastuzumab demonstrated significant reduction in tumor volume compared to vehicle, Trastuzumab alone, and the positive control Enhertu groups. In addition, PT-CD16VS infiltration into the tumor was shown to be Trastuzumab dependent, with infiltrating cells expressing high levels of CD16 and Ki67. Conclusions: Our results show that PT-CD16VS has robust in vitro and in vivo ADCC activity against gastric cancer tumor cells when combined with Trastuzumab. Furthermore, PT-CD16VS activity is specific to tumor HER2 antigen expression and does not target low HER2 expression on normal healthy cells. PT-CD16VS is being developed in combination with various monoclonal antibodies for the treatment of multiple cancers. Citation Format: Kathy Karasiewicz-Mendez, Joseph Gleason, Kristina Tess, Mansour Djedaini, Shengchen Lin, Chenfei Huang, Shuyang He, Robert Hariri, Adrian Kilcoyne. Placental circulating T cells expressing CD16 in combination with trastuzumab demonstrate robust anti-tumor antibody-dependent cellular cytotoxicity (ADCC) against gastric cancer [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 5232.

Abstract 5230: A modified FcγRI expressing T cell (SolidT) exhibits profound anti-tumor activity followed by maturation into effector memory t cells that can be reactivated to kill a secondary tumor

Abstract We previously described a subset of cytotoxic FcγRI (CD64)-expressing T cells in tumors and inflamed tissues (PMID 31449054). We subsequently expressed an engineered modified CD64 into T cells (“SolidT”) and demonstrated that these cells possessed greater specificity for tumor over normal tissues, reduced exhaustion, and attenuated cytokine secretion compared to other engineered cell therapies. SolidT activation correlates with surface tumor antigen density enabling the differentiation between tumor cells and normal cells by use of a specific antibody targeting tumor antigens. We previously showed that SolidT + mAb has anti-tumor activity in vitro and in vivo in several tumor models, including immunodeficient HER2 xenografts and an immunocompetent syngeneic B16-TYRP1 model (PMID 37070661). In this study, we further assessed the effect of SolidT on HER2+ tumors in a rechallenge model and extended the platform to CD20+ lymphoma models by combining SolidT with rituximab. In an in vivo experiment using HER2+, NCI-N87 cells in immunodeficient mice, several mice in the SolidT + trastuzumab group experienced complete tumor clearance. These mice were monitored until day 84, then rechallenged with NCI-N87 cells along with a new control group. After tumor establishment, mice were treated only with trastuzumab weekly for two weeks. While tumors in the control group progressed, the tumors in the rechallenged mice were completely cleared with a trastuzumab boost only, suggesting a SolidT cell memory response. Bone marrow from these rechallenged mice was analyzed and revealed the presence of effector memory SolidT cells. The SolidT model was then studied in a human, CD20+ lymphoma model in vitro and in vivo in immunodeficient mice. SolidT + rituximab was more potent in-vivo against CD20+ lymphoma cells than rituximab. In vivo, mice treated with SolidT + rituximab had significantly greater survival, lower tumor burden, and fewer sites of metastases than mice treated with rituximab alone. Bone marrow from these mice was analyzed and revealed the presence of effector memory SolidT cells in mice treated with SolidT + rituximab. We demonstrated that marrow resident SolidT cells developed an effector memory phenotype that positively correlated with outcome in HER2+ and CD20+ xenograft models, consistent with earlier data from a syngeneic B16 melanoma model. We further demonstrated that SolidT cells can be reactivated in a rechallenge model with only the addition of tumor targeting mAb, resulting in the eradication of the recurring tumors. This data further confirms that the SolidT platform can be targeted against different tumor antigens by using different tumor targeting mAbs. The SolidT platform could represent a paradigm shift in the approach to engineered cellular therapy for hematological malignancies and solid tumors. Citation Format: Shahar Dotan, Noam Pilpel, Peleg Rider, Hana Shpilt, Diana Rasoulouniriana, Yaron Carmi, James E. Wooldridge. A modified FcγRI expressing T cell (SolidT) exhibits profound anti-tumor activity followed by maturation into effector memory t cells that can be reactivated to kill a secondary tumor [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 5230.

Abstract 4113: AST-301,a pDNA-based cancer vaccine encoding HER2-ICD, enhances anti-tumor effect of HER2-ADC in a HER2-expressed gastric cancer xenograft model

Abstract Background: In gastric cancer, the HER2 overexpression (6% to 29.5% of gastric cancers) is considered to increase proliferation activity and suppress apoptosis of the cancer cells. However, HER2-targeting therapy in gastric cancer has not been fully evaluated. Currently, new approaches with HER2-tageted antibody-drug conjugates (ADCs) have led to promising results in HER2-related solid tumors. ADCs may have immune-modulating properties and that the payloads delivered by ADCs may induce immunogenic cell death. Based on this rationale, ADC and cancer vaccine combination would be potentially synergistic to improve clinical outcomes. This study was conducted to evaluate an synergistic effect and immune response of the combination of AST-301 and HER2-ADC in HER2-expressed gastric cancer xenograft model. AST-301 phase 1 study (PN109) was completed and phase 2 randomized-control trials (CornerStone-001, CornerStone-003) have been conducted in breast cancer and gastric cancer. Methods: NCI-N87 cells were mixed with the same amount of matrigel at 1x107 cells to formation tumors in the flanks of athymic BALB/c nude mice. After the tumor size of mice reached 100-200 mm3, all of them were randomly classified into four groups (n=7/group) and drug administration was initiated. AST-301 (100 μg/animal, intradermal injection, mixed with rhuGM-CSF as an immune adjuvant) was administered once a week to a total of fourth. LCB01 (1 mg/kg, intravenous injection) was administered to once. Enhertu (1 or 3 mg/kg, intravenous injection) was administered to twice for four weeks. The relative tumor size and TGI rate at day 25 were key endpoints of efficacy. To evaluate the mechanism underlying the enhanced anti-tumor effect due to the combined administration of AST-301 and HER2-ADCs, immune-cell profiling was also conducted using FACS analysis. Results: Our previous study has shown that AST-301 has anti-tumor effects in human gastric cancer xenograft athymic BALB/c nude mouse model. It was showed that adding AST-301 to LCB01 or Enhertu inhibited tumor growth based on a TGI rate at days 25; AST-301 combining with LCB01 vs LCB01 mono (52 % vs 43 %), AST-301 combining with Enhertu vs Enhertu mono (42 % vs 24 %).As an explorative endpoint, it was observed that myeloid-derived suppressor cells (MDSCs) elevation was modestly inhibited in tumor site in AST-301 mono or certain combination regimen adding AST-301. It was remarkable (or observed or captured) that MDSCs elevation was inhibited in tumor site in the combination groups of all ADCs with AST-301 compared to normal saline groups. Conclusion: With combining AST-301 and HER2 ADCs, it was demonstrated that anti-tumor effect was likely synergistic in the athymic mouse model. Phase 2 study of AST-301 in HER2-expressed gastric cancer has been on going (CornerStone-003, NCT 05771584). Citation Format: Jinho Kang, Hyo-Hyun Park, Jee Hyun Choi, Jinback Lim, Seong-Yong Jang, Min-Ah Kim, Myeong-Kyu Park, Yun-Hee Park, Soyeon Lim, Chul-Woong Chung, Jin Kyeong Choi, Eunkyo Joung, Ashley Yongmin Kim, Hun Jung. AST-301,a pDNA-based cancer vaccine encoding HER2-ICD, enhances anti-tumor effect of HER2-ADC in a HER2-expressed gastric cancer xenograft model [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 4113.

Abstract 2056: Click chemistry enabled selective activation of highly cytotoxic MMAE payload at tumors using TROP2 targeting agent

Abstract Shasqi is advancing the Click Activated Protodrugs Against Cancer (CAPAC®) platform based on click chemistry, a Nobel Prize winning technology. The platform is modular and comprises 1) an activator that target specific antigens, and 2) inert cancer drugs, protodrugs, which are selectively activated at tumors via click chemistry. The CAPAC technology separates the tumor targeting function from the payload and reunite them at the tumor creating the flexibility to optimize activity while limiting toxicity during preclinical and clinical development. The modularity of the platform enables the rapid development of new therapies as well as unlocking unique treatment benefits such as tunable combinations and payload cycling. We envision that CAPAC will expand the scope of potential targets, widen the therapeutic index of antibody drug conjugates (ADCs). Here we present a TROP2-targeting activator with a monomethyl auristatin E (MMAE) payload. TROP2 expression has been observed in many cancers that express low-HER2. The clinical efficacy of anti-TROP2 ADCs is hindered by undesirable side effects, limiting their potential therapeutic window. We have developed the targeting activator SQT02, a TROP2 Fab binder conjugated with ~2.2 tetrazines per protein, and SQP22, an attenuated protodrug of MMAE coupled with trans-cyclooctene (TCO). The reaction between tetrazine and TCO moieties releases MMAE at the tumor site. Previously we have demonstrated that SQP22 is attenuated compared to free MMAE and in combination with a HER2 targeting activator led to significant tumor inhibition with no observable toxicity. The CAPAC platform’s modular nature allows seamless exchange of targeting agents or payloads, thus enabling different therapeutic combinations to be generated quickly and efficiently, like SQT02 in combination with SQP22. Dosing of SQT02 occurs prior to dosing with SQP22 to reduce non-specific activation while enabling higher payload activation at the tumor site to induce tumor cell death. In vitro, SQT02 bound NCI-N87 human gastric cancer cells with similar EC50 (~0.07 μg/mL) to an antibody that binds to TROP2. In vivo, treatment of SQT02 followed by SQP22 led to 60% tumor growth inhibition compared to Sacituzumab govitecan's 35% inhibition in the NCI-N87 xenograft model. No body weight loss was observed in the animals treated with SQT02 in combination with SQP22. We have demonstrated the use of click chemistry to deliver a highly potent cytotoxic agent to tumors, causing significant growth inhibition. The flexibility and adaptability of the CAPAC platform allowed us to quickly develop therapeutic combinations that can be deployed to accelerate the path to the clinic. Our findings provide evidence for developing SQT02 in combination with SQP22 for the treatment of TROP2 expressing cancers. Citation Format: George Coricor, Jesse M. McFarland, Sangeetha Srinivasan, Stefanie Wagner, Tri-Hung Nguyen, Jose M. Mejia Oneto. Click chemistry enabled selective activation of highly cytotoxic MMAE payload at tumors using TROP2 targeting agent [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 2056.

Abstract 1913: KD5005: HER-2 dependent strong potent anti-tumor activity of an anti-HER-2 mAb-TNFaimmunocytokine

Abstract Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with diverse biological functions, including apoptosis, cell survival, inflammation, and immunity. While recombinant human TNFα has demonstrated therapeutic efficacy in isolated limb perfusion for soft tissue sarcoma and melanoma, its systemic administration is limited by severe toxicities, including hypotension. Tumor-specific targeting and enhancement of TNFα's tumor cell killing activity hold promise for improving its therapeutic index. To address these challenges, we developed KD5005, a novel HER-2-targeted immunocytokine fusion protein comprising an anti-HER-2 IgG1 monoclonal antibody (trastuzumab) and human TNFα. Tissue distribution studies revealed that KD5005 specifically accumulated in HER-2-expressing tumors, demonstrating its tumor-targeting capability. In vitro, KD5005 exhibited reduced TNFα-mediated apoptosis in L929 cells compared to recombinant TNFα, indicating its lower non-specific toxicity. Notably, KD5005 induced apoptosis of HER-2-positive human gastric cancer NCI-N87 cells at significantly lower concentrations than trastuzumab, TNFα, or their combination, demonstrating its enhanced tumor cell killing activity. The potent apoptotic activity of KD5005 was HER-2-dependent, as it did not show increased apoptotic activity in WT mouse forestomach carcinoma (MFC) cells and had similar apoptotic activity compared to the combination of trastuzumab and TNFα. This HER-2 dependency further supports the tumor-specific targeting of KD5005.In vivo, KD5005 demonstrated superior anti-tumor efficacy compared to the combination of trastuzumab and TNFα in syngeneic tumor models using CT26 tumor cells transfected with the human HER-2 gene. KD5005 induced robust tumor regression and durable cure in 7 of 8 mice, while the combination of trastuzumab and TNFα elicited weaker therapeutic responses, resulting in tumor rejection in only 3 of 8 mice. Pharmacokinetic and toxicity studies of KD5005 were conducted in cynomolgus monkeys. KD5005 exhibited a significantly prolonged in vivo half-life compared to TNFα, indicating its improved pharmacokinetic profile. KD5005 was administered intravenously every two weeks to the same animal in a dose-escalation study at 0.3 mg/kg, 1.0 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg. The toxicity study results showed that the maximum tolerated dose for KD5005 in cynomolgus monkeys is 10 mg/kg, indicating its favorable toxicity profile. In summary, we have developed KD5005, a HER-2-targeted immunocytokine fusion protein with enhanced tumor-killing activity, improved pharmacokinetic properties, and a favorable toxicity profile. These preclinical findings support the further clinical development of KD5005 as a promising therapeutic agent for HER-2-positive cancers. Citation Format: YI CHEN, PENG LI, GUOBO FANG, HENG WU, CHI ZHANG, ZELING CAI. KD5005: HER-2 dependent strong potent anti-tumor activity of an anti-HER-2 mAb-TNFaimmunocytokine [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 1913.

Abstract 6581: DX126-262 combined with chemotherapy (Cisplatin and 5-Fu) demonstrates promising antitumor efficacy in HER2-positive gastric cancer

Abstract Gastric cancer is a common malignant tumor of the digestive system, and its morbidity and mortality rank among the top five in the world. Although significant progress has been made in cytotoxic chemotherapy, immunotherapy and targeted therapy in recent years, the long-term prognosis of patients with advanced or metastatic gastric cancer is still poor. Trastuzumab combined with chemotherapy is the first-line standard of care (SOC) for HER2-positive advanced or metastatic gastric cancer. Two ADC drugs targeting HER2, DS-8201 and RC-48, have also been approved for the treatment of gastric cancer. However, their limited clinical benefits and occurrence of adverse events still present a significant challenge. Therefore, minimizing the off-target toxicity and collateral damage while achieving significant chemotherapy efficacy has become the focus of gastric cancer treatment. DX126-262 is a novel HER2-targeting antibody-drug conjugate (ADC), generated by conjugating a Tubulysin B analogue to a recombinant humanized anti-HER2 monoclonal antibody through a linker containing branched hydrophilic polyethylene glycol, using thiol-maleimide chemistry to achieve drug antibody ratio (DAR) of 3.8. Previous studies have confirmed that DX126-262 monotherapy showed excellent efficacy in HER2-positive gastric cancer NCI-N87 cells in vitro and in vivo. And the efficacy has also been demonstrated in clinical trials (now in phase II, CTR20211871). Based on above results, we intended to further explore whether combination therapy of DX126-262 with Cisplatin and 5-FU can improve the anti-tumor efficacy compared with SOC (Herceptin plus Cisplatin and 5-FU) or DS-8201 monotherapy. The triple-drug combination therapy demonstrated much better therapeutic efficacy than single drug (DX126-262, Cisplatin, 5-FU, Herceptin, or DS-8201) or double-drug combination (Cisplatin plus 5-FU) or SOC (Herceptin plus Cisplatin and 5-FU) on human gastric cancer cells in vitro and in vivo. Meanwhile, triple-drug combination therapy did not exhibit superimposed toxicity, judging by the body weight of mice and hemal biochemistry assays. These results suggested that DX126-262 plus Cisplatin and 5-FU might be a promising strategy for treatment of HER2-positive advanced or metastatic gastric cancer in clinic. Citation Format: Xiaobo Cai, Min Cao, Huihui Guo, Qingliang Yang, Xiangfei Kong, Yong Du, Zhicang Ye, Zhixiang Guo, Lingli Zhang, Lu Bai, Junxiang Jia, Yunxia Zheng, Yongxiang Chen, Miaomiao Chen, Wei Zheng, Jun Zheng, Wenjun Li, Yuanyuan Huang, Mengmeng Liu, Zhongliang Fan, Hangbo Ye, Yifang Xu, Binbin Chen, Meng Dai, Robert Y. Zhao. DX126-262 combined with chemotherapy (Cisplatin and 5-Fu) demonstrates promising antitumor efficacy in HER2-positive gastric cancer [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 6581.

A Cell-Penetrating Peptide Improves Anti-HER2 Single-Chain Variable Fragment Internalization and Antitumor Activity against HER2-Positive Breast Cancer In Vitro and In Vivo.

Cell-penetrating peptides (CPPs) are invaluable tools for delivering various substances into cells by crossing biological membranes. However, the effects of cell-penetrating peptide fusion proteins on the biological activity of antibodies remain to be fully understood. Here, we engineered a recombinant protein, LP-scFv, which combines the single-chain variable region of anti-human epidermal growth factor receptor-2 with a novel and non-oxic cell-penetrating peptide as a leader peptide. The introduction of this leader peptide led to a more than twofold increase in the internalization efficiency of the single-chain antibody, as confirmed using microscopic analysis and flow cytometry. The effects of the single-chain antibodies and LP-scFv on cell viability were evaluated using the MTT assay. Both the single-chain antibodies and LP-scFv reduced the viability of BT474 and NCI-N87 cells in a dose-dependent manner while exhibiting minimal toxicity towards MCF-7 and MCF-10A cells. Further investigation into LP-scFv's mechanism revealed that the induced leader peptide does not alter the MAPK-ERK1/2 and PI3K/AKT pathways of single-chain antibodies. An enhanced antitumor activity was also confirmed in an NCI-N87 tumor xenograft model in mice with a reduction of 45.2% in tumor growth inhibition (vs. 23.1% for scFv) with a 50 mg/kg dose after orthotopic injection administration, which was equivalent to that of trastuzumab (vs. 55.7% for trastuzumab). Overall, these results indicate that LP-scFv exhibits significant permeation activity in HER2-positive cells to enhance the intracellular dose effect on antitumor activity in vitro and in vivo. This research lays the foundation for designing novel antibody-based therapies for cancer.

Abnormal activation of RFC3, A YAP1/TEAD downstream target, promotes gastric cancer progression.

Gastric cancer (GC) is a malignant tumor with a high mortality rate, and thus, it is necessary to explore molecular mechanisms underlying its progression. While replication factor C subunit 3 (RFC3) has been demonstrated to function as an oncogene in many cancers, its role in GC remains unclear. Tumor tissues were collected from clinical GC patients, and the expression of RFC3 was analyzed. NCI-N87 and HGC-27 cells were infected with lentivirus sh-RFC3 to knock down RFC3 expression. RFC3 expression levels were determined, in addition to cell biological behaviors both in vitro and in vivo. The relationship between RFC3 and the YAP1/TEAD signaling pathway was detected by dual luciferase reporter assay. RFC3 was upregulated in GC tumor tissues. RFC3 knockdown inhibited cell proliferation, promoted cell apoptosis of GC cells, and suppressed cell migration and invasion. Moreover, depleted RFC3 suppressed tumor growth and metastasis in vivo. Mechanistically, the YAP1/TEAD axis activated RFC3 expression transcriptionally by binding to the RFC3 promoter. RFC3 was transcriptional activated by the YAP1/TEAD signaling pathway, thus promoting GC progression. RFC3 may be a promising therapeutic target for GC.

Tribbles Genes in Gastric Cancer: A Tumor-Suppressive Role for TRIB2.

Tribbles pseudokinases (TRIB1-3) are important signaling modulators involved in several cancers. However, their function in gastric cancer (GC) remains undefined. GC is still a deadly disease since the lack of sensitive and specific biomarkers for early diagnosis and therapy response prediction negatively affects patients' outcome. The identification of novel molecular players may lead to more effective diagnostic and therapeutic avenues. Therefore, we investigated the role of TRIB genes in gastric tumorigenesis. Data mining of the TCGA dataset revealed that chromosomal instability (CIN) tumors have lower TRIB2 and higher TRIB3 expression versus microsatellite instability (MSI)-high tumors, while TRIB1 levels are similar in both tumor types. Moreover, in CIN tumors, low TRIB2 expression is significantly associated with aggressive stage IV disease. As no studies on TRIB2 in GC are available, we focused on this gene for further in vitro analyses. We checked the effect of TRIB2 overexpression (OE) on MKN45 and NCI-N87 CIN GC cell lines. In MKN45 cells, TRIB2 OE reduced proliferation and colony formation ability and induced G2/M arrest, while it decreased the proliferation and cell motility of NCI-N87 cells. These effects were not mediated by the MAPK pathway. Our results suggest a tumor-suppressive function of TRIB2 in GC with a CIN phenotype.

MicroRNAs as biomarkers for trastuzumab-based therapy in HER2-positive advanced oesophago-gastric cancer patients

BackgroundThis study aimed to identify microRNAs (miRs) as circulating biomarkers of resistance to first-line trastuzumab-based therapy in advanced HER2-positive oesophago-gastric cancer patients.MethodsA high-throughput 1015 Exiqon miRCURY LNA™ microRNA inhibitor library screen was performed in trastuzumab-treated HER2-positive NCI-N87 and HER2-negative FLO-1 oesophago-gastric cancer cell lines. NanoString nCounter® miR analysis was performed in NCI-N87, FLO-1, and MAGIC trial (ISRCTN93793971) formalin-fixed paraffin-embedded (FFPE) oesophago-gastric cancer patient samples. MiR-148a-3p copies in plasma samples were quantified using digital droplet polymerase chain reaction (ddPCR) from HER2-positive oesophago-gastric cancer patients treated with standard-of-care trastuzumab-based therapy within the FOrMAT (NCT02112357) and PLATFORM (NCT02678182) clinical trials. The primary endpoints were overall survival (OS) for plasma miR-148a-3p HIGH (>median) versus LOW (≤median). The secondary endpoints were progression-free survival (PFS) and 3-month progression-free rates (PFRs) miR-148a-3p HIGH versus LOW. PLATFORM sensitivity analysis normalised miR-148a-3p (NmiR-148a-3p).ResultsThe inhibition of miR-148a-3p reduced NCI-N87 relative cell viability (<0.6) and expression was high (>242) in NCI-N87 and HER2-positive MAGIC trial patients (n=5). Normalised-miR-148a-3p (NmiR-148a-3p) LOW versus HIGH demonstrated a statistically significant difference in 3-month PFRs (n=23; OR, 0.11 [0.02–0.78]; p=0.027; aOR, 0.03 [0.001–0.71], p=0.029) but no difference in OS or PFS. There was no statistically significant relationship between miR-148-3p LOW versus HIGH for OS (PLATFORM, n=62; hazard ratio [HR], 0.98 [0.57–1.66]; p=0.933; FOrMAT, n=8; HR, 0.54 [0.13–2.31]; p=0.322), PFS (n=62; HR, 1.08 [0.65–1.81]; p=0.759; FOrMAT, n=8; HR, 1.26 [0.31–5.07]; p=0.714), or PFRs (PLATFORM, n=31; odds ratio [OR], 0.67 [0.2–2.8]; p=0.577).ConclusionNormalised miR-148a-3p may be a relevant biomarker for trastuzumab-based therapy in advanced HER2-positive oesophago-gastric cancer patients.

Discovery of potent STAT3 inhibitors using structure-based virtual screening, molecular dynamic simulation, and biological evaluation.

Signal transducer and activator of transcription 3 (STAT3) is ubiquitously hyper-activated in numerous cancers, rendering it an appealing target for therapeutic intervention. In this study, using structure-based virtual screening complemented by molecular dynamics simulations, we identified ten potential STAT3 inhibitors. The simulations pinpointed compounds 8, 9, and 10 as forming distinct hydrogen bonds with the SH2 domain of STAT3. In vitro cytotoxicity assays highlighted compound 4 as a potent inhibitor of gastric cancer cell proliferation across MGC803, KATO III, and NCI-N87 cell lines. Further cellular assays substantiated the ability of compound 4 to attenuate IL-6-mediated STAT3 phosphorylation at Tyr475. Additionally, oxygen consumption rate assays corroborated compound 4's deleterious effects on mitochondrial function. Collectively, our findings position compound 4 as a promising lead candidate warranting further exploration in the development of anti-gastric cancer therapeutics.

Gefitinib Induces Apoptosis of Gastric Cancer Cells by Promoting Glutaminolysis Through the MEK/ERK Signaling Pathway

We aimed to investigate the effect of gefitinib on the proliferation and apoptosis of gastric cancer (GC) cells through the MEK/ERK signaling pathway. The GC cell line NCI-N87 was cultured in vitro and divided into a control group and a gefitinib group. Cell viability for NCI-N87 was determined using the MTT test. The amount of apoptosis in NCI-N87 cells was measured using flow cytometry. Real-time quantitative PCR was used to measure GDH1 mRNA expression in NCI-N87 cells. P-MEK1/2, MEK1/2, P-ERK1/2, and ERK1/2 protein expression levels in NCI-N87 cells were determined using Western blotting. An assay kit for measuring glutamine was used to determine the intracellular glutamine concentration of NCI-N87 cells. The proliferative activity of NCI-N87 cells was significantly inhibited in the gefitinib group compared to the control group, along with the transcription level of GDH1, intracellular glutamine content, intracellular glutamine content reduction, and p-MEK1/2 and p-ERK1/2 protein expression levels. Gefitinib inhibits proliferation and promotes apoptosis in the GC cell line NCI-N87 by downregulating the EGFR/MEK/ERK signaling pathway. Mechanistically, it is achieved by promoting the expression of GDH1 to promote glutaminolysis in NCI-N87 cells.

Lysine-specific Demethylase 6A Upregulates Cadherin-1 and Accelerates Gastric Cancer Growth.

Gastric cancer (GC) ranks fifth among all common malignancies globally. Genetic research has revealed several genes that are frequently dis-regulated in GC, such as lysine-specific demethylase 6A (KDM6A) and cadherin-1 (CDH1). This study aimed to examine the expression profile and role of KDM6A in GC, as well as the molecular pathway involved. The expression profile and overall survival data of KDM6A were retrieved from the TCGA database. Expression levels of KDM6A were also measured in GC patient samples and compared with those of healthy controls. Furthermore, stable silencing of KDM6A was introduced into the GC cell line NCI-N87, followed by assessments of cell proliferation, migration and invasion, in the xenograft mouse model. The metastatic status of mice injected with NCI-N87 cells was also analyzed. In patients diagnosed with GC, KDM6A was upregulated. Silencing KDM6A reduced the proliferation, migration and invasion of cells, as well as the growth of xenograft tumors. KDM6A knockdown also inhibited metastatic behaviors of injected NCI-N87 cells, as well as elevated CDH1 expression, leading to reversed epithelial-mesenchymal transition. KDM6A serves as an oncogene in GC and exerts its pro-tumor functions by repressing the expression of CDH1.