Preclinical Gastric Cancer Models Research Articles

Thioridazine reverses trastuzumab resistance in gastric cancer by inhibiting S-phase kinase associated protein 2-mediated aerobic glycolysis.

Trastuzumab constitutes the fundamental component of initial therapy for patients with advanced human epidermal growth factor receptor 2 (HER-2)-positive gastric cancer (GC). However, the efficacy of this treatment is hindered by substantial challenges associated with both primary and acquired drug resistance. While S-phase kinase associated protein 2 (Skp2) overexpression has been implicated in the malignant progression of GC, its role in regulating trastuzumab resistance in this context remains uncertain. Despite the numerous studies investigating Skp2 inhibitors among small molecule compounds and natural products, there has been a lack of successful commercialization of drugs specifically targeting Skp2. To discover a Skp2 blocker among currently available medications and develop a therapeutic strategy for HER2-positive GC patients who have experienced progression following trastuzumab-based treatment. Skp2 exogenous overexpression plasmids and small interfering RNA vectors were utilized to investigate the correlation between Skp2 expression and trastuzumab resistance in GC cells. Q-PCR, western blot, and immunohistochemical analyses were conducted to evaluate the regulatory effect of thioridazine on Skp2 expression. A cell counting kit-8 assay, flow cytometry, a amplex red glucose/glucose oxidase assay kit, and a lactate assay kit were utilized to measure the proliferation, apoptosis, and glycolytic activity of GC cells in vitro. A xenograft model established with human GC in nude mice was used to assess thioridazine's effectiveness in vivo. The expression of Skp2 exhibited a negative correlation with the sensitivity of HER2-positive GC cells to trastuzumab. Thioridazine demonstrated the ability to directly bind to Skp2, resulting in a reduction in Skp2 expression at both the transcriptional and translational levels. Moreover, thioridazine effectively inhibited cell proliferation, exhibited antiapoptotic properties, and decreased the glucose uptake rate and lactate production by suppressing Skp2/protein kinase B/mammalian target of rapamycin/glucose transporter type 1 signaling pathways. The combination of thioridazine with either trastuzumab or lapatinib exhibited a more pronounced anticancer effect in vivo, surpassing the efficacy of either monotherapy. Thioridazine demonstrates promising outcomes in preclinical GC models and offers a novel therapeutic approach for addressing trastuzumab resistance, particularly when used in conjunction with lapatinib. This compound has potential benefits for patients with Skp2-proficient tumors.

P70S6K/Akt dual inhibitor DIACC3010 is efficacious in preclinical models of gastric cancer alone and in combination with trastuzumab

The PI3K-Akt-mTOR (PAM) pathway is implicated in tumor progression in many tumor types, including metastatic gastric cancer (GC). The initial promise of PAM inhibitors has been unrealized in the clinic, presumably due, in part, to the up-regulation of Akt signaling that occurs when the pathway is inhibited. Here we present that DIACC3010 (formerly M2698), an inhibitor of two nodes in the PAM pathway, p70S6K and Akt 1/3, blocks the pathway in in vitro and in vivo preclinical models of GC while providing a mechanism that inhibits signaling from subsequent Akt up-regulation. Utilizing GC cell lines and xenograft models, we identified potential markers of DIACC3010-sensitivity in Her2-negative tumors, i.e., PIK3CA mutations, low basal pERK, and a group of differentially expressed genes (DEGs). The combination of DIACC3010 and trastuzumab was evaluated in Her2-positive cell lines and models. Potential biomarkers for the synergistic efficacy of the combination of DIACC3010 + trastuzumab also included DEGs as well as a lack of up-regulation of pERK. Of 27 GC patient-derived xenograft (PDX) models tested in BALB/c nu/nu mice, 59% were sensitive to DIACC3010 + trastuzumab. Of the 21 HER2-negative PDX models, DIACC3010 significantly inhibited the growth of 38%. Altogether, these results provide a path forward to validate the potential biomarkers of DIACC3010 sensitivity in GC and support clinical evaluation of DIACC3010 monotherapy and combination with trastuzumab in patients with HER2- negative and positive advanced GCs, respectively.

The BRD4 inhibitor JQ1 augments the antitumor efficacy of abemaciclib in preclinical models of gastric carcinoma

BackgroundAdvanced gastric cancer (GC) is a lethal malignancy, harboring recurrent alterations in cell cycle pathway, especially the CDKN2A-CDK4/CDK6/CCND1 axis. However, monotherapy of CDK4/6 inhibitors has shown limited antitumor effects for GC, and combination treatments were urgently needed for CDK4/6 inhibitors.MethodsHere, we performed a comprehensive analysis, including drug screening, pan-cancer genomic dependency analysis, and epigenetic sequencing to identify the candidate combination with CDK4/6 inhibitors. Mechanisms were investigated by bulk RNA-sequencing and experimental validation was conducted on diverse in vitro or in vivo preclinical GC models.ResultsWe found that the BRD4 inhibitor JQ1 augments the antitumor efficacy of the CDK4/6 inhibitor abemaciclib (ABE). Diverse in vitro and in vivo preclinical GC models are examined and synergistic benefits from the combination therapy are obtained consistently. Mechanistically, the combination of ABE and JQ1 enhances the cell cycle arrest of GC cells and induces unique characteristics of cellular senescence through the induction of DNA damage, which is revealed by transcriptomic profiling and further validated by substantial in vitro and in vivo GC models.ConclusionThis study thus proposes a candidate combination therapy of ABE and JQ1 to improve the therapeutic efficacy and worth further investigation in clinical trials for GC.

SMG8/SMG9 Heterodimer Loss Modulates SMG1 Kinase to Drive ATR Inhibitor Resistance.

These findings reveal how cancer cells acquire resistance to ATRi and identify pathways that could be targeted to enhance the overall effectiveness of these inhibitors.

Abstract 1069: OBT076, a clinical-stage ADC, displays synergy with oxaliplatin and cisplatin in preclinical gastric cancer models

Abstract CD205 is a transmembrane glycoprotein, robustly expressed in malignancies from varied histotypes, making it an ideal target for Antibody Drug Conjugate (ADC) therapy. OBT076 is a novel and selective clinical stage ADC with potent activity against CD205-positive liquid and solid tumors. We explored the cellular cytotoxicity profile of combination chemotherapy with OBT076 and two standard-of-care platinum-based compounds, Oxaliplatin (Ox) and Cisplatin (Cis), in preclinical gastric, pancreatic and colorectal cancer models. A panel of gastric cancer and other solid tumor cell lines were selected, based on mRNA data, for CD205 expression. This was confirmed using flow cytometry. The cell lines were assessed for susceptibility to monotherapy with OBT076, Ox and Cis. Cells lines displaying positive cytotoxic responses were assessed for susceptibility to combination chemotherapy by first treating cells with a single dose of OBT076 for 72 hours, leading to 20% growth inhibition (IC20), and subsequently with either Ox or Cis for 48 hours (OBT-Ox and OBT-CIS, respectively). The ATP-dependent fluorescence-based cellular viability system cell titer-glo was used as the cytotoxic readout. In our preclinical gastric cancer model, pre-treatment with OBT076 followed by either Ox or Cis displayed synergistic combination effects, reducing the half-maximal inhibitory concentration (IC50) of Ox and Cis by 40- and 10-fold respectively. The resultant cellular cytotoxicity of OBT-Ox and OBT-Cis combination therapy was higher compared to monotherapy with either agent. Moreover, while monotherapy with Ox was less potent than with Cis, OBT-Ox achieved similar potency to OBT076-Cis. For colorectal and pancreatic cancer models, the effects were less pronounced: OBT076-combination therapy increased Platinum sensitivity by 2 to 5 times. No synergistic effects were observed when the treatment order was reversed. These results and those from ongoing in vivo studies in gastric cancer models will be discussed. In summary OBT076, a clinical stage ADC displays synergistic anti-tumor effects in preclinical gastric cancer models, when used in combination with Ox or Cis. OBT076 is currently being evaluated in the gastric clinical population where Platinum-based therapies remain the mainstay of treatment. Citation Format: Arnima Bisht, Murray Cox, Chander Sekhar Peddaboina, Michael Crocamo, Abderrahim Fandi, Christian Rohlff. OBT076, a clinical-stage ADC, displays synergy with oxaliplatin and cisplatin in preclinical gastric cancer models [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 1069.

Targeted FGFR/VEGFR/PDGFR inhibition with dovitinib enhances the effects of nab-paclitaxel in preclinical gastric cancer models

ABSTRACT Standard chemotherapy regimens for gastric adenocarcinoma (GAC) have limited efficacy and considerable toxicity profiles. Nab-paclitaxel has shown promising antitumor benefits in previous GAC preclinical studies. Dovitinib inhibits members of the receptor tyrosine kinase family including FGFR, VEGFR and PDGFR, and has exhibited antitumor effects in many solid tumors including GAC. Based on the antimitotic, antistromal and EPR effects of nab-paclitaxel, we investigated augmentation of nab-paclitaxel response by dovitinib in multiple GAC preclinical models. In MKN-45 subcutaneous xenografts, inhibition in tumor growth by nab-paclitaxel and dovitinib was 75% and 76%, respectively. Dovitinib plus nab-paclitaxel had an additive effect on tumor growth inhibition and resulted in tumor regression (85% of its original value). Dovitinib monotherapy resulted in minimal improvement in animal survival (25 days) compared to control (23 days), while nab-paclitaxel monotherapy or dovitinib plus nab-paclitaxel combination therapy led to a clinically significant lifespan extension of 83% (42 days) and 187% (66 days), respectively. IHC analysis of subcutaneous tumors exhibited reduced tumor cell proliferation and tumor vasculature by dovitinib. In vitro studies demonstrated that dovitinib and nab-paclitaxel individually reduced tumor cell proliferation, with an additive effect from combination therapy. Immunoblot analyses of MKN-45 and KATO-III cells revealed that dovitinib decreased phospho-FGFR, phospho-AKT, phospho-ERK, phospho-p70S6K, phospho-4EBP1, Bcl-2 and increased cleaved PARP-1, cleaved-caspase-3, p27, Bax, Bim, with an additive effect from combination therapy. These results demonstrate that the FGFR/VEGFR/PDGFR inhibitor, dovitinib, has the potential to augment the antitumor effects of nab-paclitaxel, with implications for use in the advancement of clinical GAC therapy.

P16 methylation is a potential predictive marker for abemaciclib sensitivity in gastric cancer

Cell cycle control is often disrupted in gastric cancer (GC), making it an attractive therapeutic target. Abemaciclib is a specific CDK4/6 inhibitor that has been shown to improve treatment efficacy in hormone receptor-positive advanced breast cancer; however, its potential therapeutic value and predictive markers have not yet been revealed in GC. In this study, we investigated the efficacy of abemaciclib using preclinical GC models representing defined molecular subtypes from The Cancer Genome Atlas. In these 49 GC cell lines, Epstein–Barr virus (EBV) and high microsatellite instability (MSI-H)-type cell lines were p16 methylated and sensitive to abemaciclib; further, genomically stable (GS), and chromosomal instability (CIN)-type cell lines with p16 methylation and intact Rb were also found to be responsive. In addition, we found that GC patients with p16 methylation often displayed a poor prognosis. Collectively, these data provide a foundation for clinical trials to assess the therapeutic efficacy of abemaciclib in GC and suggest that p16 methylation could be used as a predictive marker to identify patients with GC who may benefit from abemaciclib-based therapies.

Abstract 553: Antitumor efficacy of a liposomal formulation of irinotecan in preclinical gastric cancer models: Augmenting its response by antiangiogenic agents

Abstract BACKGROUND: Gastric adenocarcinoma (GAC) remains the 3rd most common cause of cancer-related deaths worldwide. Systemic chemotherapy is commonly a fundamental treatment for metastatic GAC that usually leads to a modest patient benefit, resulting in a 5-year survival rate of 31%. A liposomal formulation of irinotecan (nal-IRI) has shown improved pharmacokinetic and drug biodistribution compared with free irinotecan (IRI) in preclinical studies. Angiogenesis plays a crucial role in the progression and metastasis of GAC. We evaluated the therapeutic efficacy of nal-IRI in combination with nintedanib, a tyrosine kinase inhibitor targeting FGFR, PDGFR and VEGFR, and DC101, a monoclonal antibody targeting VEGFR2 in preclinical models of GAC. METHODS: In vitro cell proliferation was evaluated in three GAC cell lines (MKN-45, KATO-III and SNU-5) by WST-1 assay. Protein expression was measured by Western blot analysis in MKN-45 cell lysates. Animal survival studies were performed using two cell lines MKN-45 and KATO-III cells in peritoneal dissemination models in NOD/SCID mice (n=5-7). Tumor growth and pharmacokinetic studies were performed in GAC cell-derived xenografts. Mechanistic evaluation included IHC and Immunoblot analysis in tumor tissues. RESULTS: Animal survival was increased by nal-IRI (>156%) and IRI (>94%) therapy compared with PBS-treated controls. Importantly, nal-IRI led to a >30% extension of animal survival compared with IRI. The addition of antiangiogenic agents nintedanib or DC101 led to a further improvement (range 3-40%) in animal survival caused by nal-IRI and IRI. In GAC cell-derived subcutaneous xenografts, compared to controls, nal-IRI demonstrated greater tumor growth inhibition (92%) than IRI (71%). Here, the combinations of nal-IRI or IRI with antiangiogenic agents exhibited an additive response. Ki67 staining of tumor sections revealed that nal-IRI was most effective in reducing intratumoral proliferation (by 58%), followed by IRI (39%), nintedanib (33%), and DC101 (25%). Combinations of nal-IRI or IRI with antiangiogenic agents demonstrated an additive effect in reducing tumor cell proliferation. As expected, tumor vasculature (assessed by endomucin staining) was reduced by nintedanib (65%) and DC101 (58%), while nal-IRI and IRI showed no effect. Pharmacokinetic studies revealed that nal-IRI increased the retention, circulation time and tumor levels of IRI and its active metabolite SN-38. The addition of nintedanib or DC101 had no effect on plasma or tumor levels of IRI or SN38. In in vitro cell viability assays in mutationally different GAC cells, SN-38 had a dose-dependent growth inhibitory effect that was more pronounced than single-agent IRI. Importantly, combinations of SN-38 or IRI with antiangiogenic agents demonstrated additive effects on cell proliferation inhibition. CONCLUSION: nal-IRI showed greater antitumor efficacy than IRI, and its antitumor effects can be enhanced by antiangiogenic agents suggesting that this combination has potential for improving clinical GAC therapy. Citation Format: Niranjan Awasthi, Margaret A. Schwarz, Changhua Zhang, Stephan Klinz, Florence Meyer-Losic, Benjamin Beaufils, Arunthathi Thiagalingam, Roderich E. Schwarz. Antitumor efficacy of a liposomal formulation of irinotecan in preclinical gastric cancer models: Augmenting its response by antiangiogenic agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 553.

PI3K-targeting strategy using alpelisib to enhance the antitumor effect of paclitaxel in human gastric cancer

PIK3CA mutations are frequently observed in various human cancers including gastric cancer (GC). This study was conducted to investigate the anti-tumor effects of alpelisib, a PI3K p110α-specific inhibitor, using preclinical models of GC. In addition, the combined effects of alpelisib and paclitaxel on GC were evaluated. Among the SNU1, SNU16, SNU484, SNU601, SNU638, SNU668, AGS, and MKN1 GC cells, three PIK3CA-mutant cells were predominantly sensitive to alpelisib. Alpelisib monotherapy decreased AKT and S6K1 phosphorylation and induced G0/G1 phase arrest regardless of PIK3CA mutational status. The alpelisib and paclitaxel combination demonstrated synergistic anti-proliferative effects, preferentially on PIK3CA-mutant cells, resulting in increased DNA damage response and apoptosis. In addition, alpelisib and paclitaxel combination potentiated anti-migratory activity in PIK3CA-mutant cells. Alpelisib partially reversed epithelial–mesenchymal transition markers in PIK3CA-mutant cells. In a xenograft model of MKN1 cells, the alpelisib and paclitaxel combination significantly enhanced anti-tumor activity by decreasing Ki-67 expression and increasing apoptosis. Moreover, this combination tended to prolong the survival of tumor-bearing mice. Our data suggest promising anti-tumor efficacy of alpelisib alone or in combination with paclitaxel in PIK3CA-mutant GC cells.

A multicenter phase II trial of tumor treating fields plus chemotherapy for first-line treatment of gastric adenocarcinoma.

TPS474 Background: Gastric carcinoma (GC) is the third-leading cause of death in China (291,000 deaths in 2015). Current therapies include surgery, chemotherapy, radiotherapy and targeted therapy, which prolong PFS and OS to 6 months and 8-14 months, respectively. Tumor Treating Fields (TTFields) are a non-invasive, regional antimitotic treatment modality approved by the FDA for glioblastoma and malignant pleural mesothelioma. TTFields at specific frequency (100- 500 kHz) are delivered via transducer arrays placed on the skin of the upper abdomen, back, right and left hypochondriac regions where the primary tumor lesion is located. TTFields were effective in preclinical models of gastric cancer and there are several ongoing Phase 3 trials of TTFields in multiple solid tumors. In this phase 2, single arm, open-label, multi-center study, we will investigate for the first time the efficacy and safety of TTFields concomitant with XELOX (oxaliplatin/capecitabine) as the first-line treatment of GC. Methods: Patients (N = 50) with histologically confirmed unresectable, locally advanced or metastatic Gastroesophageal Junction (GEJ) or Gastric Adenocarcinoma (GC), aged ≥ 18 years, ECOG PS 0-1, who had no previous systemic treatment for the recurrent or metastatic disease will be enrolled. Patients will receive TTFields (150 kHz via the NovoTTF-100L (P) medical device for average monthly use of 18 hrs/day) plus XELOX chemotherapy (Oxaliplatin: 130 mg/m2 on day 1 every 3 weeks; Capecitabine: 1000 mg/m2, PO, BID on day 1-14 every 3 weeks). For HER-2 positive patients, trastuzumab is allowed. The primary endpoint is investigator-assessed Objective Response Rate (ORR) per RECIST 1.1. Secondary endpoints are time to tumor progression (TTP), progression-free survival (PFS), overall survival (OS), and 12-month OS rate. Adverse events (AEs) will be graded for severity according to CTCAE 5.0. Based on the historical ORR data in first-line chemotherapy in GC, we assumed that ORR will be higher than 45% with TTFields concomitant with chemotherapy. At least 45 patients need to be enrolled to ensure the lower boundary is 30% of the 95% CI. Estimating a patient drop-out rate of 10%, 50 patients will be actually enrolled.

Abstract 2208: Targeted inhibition of FGF/PDGF/VEGF signaling enhances nanoparticle taxane response in preclinical gastric cancer models

Abstract Background: Gastric adenocarcinoma (GAC) remains the third most common cause of cancer-related deaths worldwide. Most available systemic chemotherapy options for advanced GAC have limited efficacy. Nab-paclitaxel (NPT), a nanoparticle albumin-bound taxane formulation, has shown significant activity in preclinical GAC studies. Overexpression of multiple growth factors and their receptors such as FGF/FGFR, PDGF/PDGFR and VEGF/VEGFR, promote angiogenesis in several cancers including GAC that leads to tumor progression and metastasis. Dovitinib is a small molecule RTK inhibitor that potentially inhibits the activity of FGFR, PDGFR and VEGFR. We evaluated the antitumor efficacy of dovitinib and its ability to enhance nab-paclitaxel response in preclinical GAC models. Methods: In vitro cell proliferation and protein expression were measured by WST-1 assay and Immunoblotting. In vivo tumor growth and animal survival studies were performed in NOD/SCID mice using human GAC MKN-45 cells. Results: Nab-paclitaxel and dovitinib had in vitro growth inhibitory effect on several GAC associated cell lines tested, with additive effects in combination. Immunoblot analysis revealed that dovitinib treatment caused a decrease in the expression of phospho-FGFR, phospho-AKT, phospho-ERK; and an increase in the expression of apoptosis-related proteins cleaved PARP-1 and cleaved caspase-3. Nab-paclitaxel activity correlated with the increased expression of phospho-stathmin. In subcutaneous GAC xenografts, NPT and dovitinib demonstrated inhibition in tumor growth, while NPT+dovitinib had an additive effect. Net tumor growth was 533.7 mm3 in controls, 394.8 mm3 after oxaliplatin, 135.8 mm3 after NPT, 128 mm3 after dovitinib, 98.9 mm3 after oxaliplatin+dovitinib and -41.8 mm3 (tumor regression) after NPT+dovitinib. Tumor tissue analysis revealed that dovitinib reduced tumor vasculature, while nab-paclitaxel reduced tumor cell proliferation. In a peritoneal dissemination model, animal survival compared to controls (23 days) remained unchanged after monotherapy with oxaliplatin (24 days) or dovitinib (25 days) but increased significantly after NPT monotherapy (42 days, a 83% increase). Combination of dovitinib with NPT exhibited a further increase in animal survival (66 days, a 187% increase), while the combination of dovitinib with oxaliplatin had no survival benefit (25 days). Conclusion: These findings demonstrate that the antitumor effect of nab-paclitaxel can be significantly enhanced by the FGFR/PDGFR/VEGFR pathway inhibitor dovitinib in preclinical GAC models. The data support the clinical relevance of this therapeutic combination for advanced GAC patients. Citation Format: Niranjan Awasthi, Kate Crawford, Erin Bontrager, Sazzad Hassan, Urs von Holzen, Margaret A. Schwarz, Roderich E. Schwarz. Targeted inhibition of FGF/PDGF/VEGF signaling enhances nanoparticle taxane response in preclinical gastric cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2208.

Abstract 1442: Chimeric antigen receptor T cell therapy targeting ICAM-1 in gastric cancer

Abstract Introduction: Despite advances in treatment, gastric cancer (GC) remains among the most fatal malignancies. Intercellular adhesion molecule-1 (ICAM-1) is overexpressed and associated with various cancers, including GC. We developed a third-generation chimeric antigen receptor (CAR) targeting ICAM-1 (ICAM-1-CAR) and investigated ICAM-1 as a immunotherapeutic target for CAR T therapy in GC. Methods: We investigated ICAM-1 expression in tissues of curatively resected GC patients (n=134) by immunohistochemical staining to determine its prognostic value. Effector to target assays were performed with ICAM-1-CAR T cells co-cultured with 8 GC cell lines with varying levels of ICAM-1 expression to investigate specific target cell death. We created a firefly luciferase-expressing human GC model in mice to measure tumor growth and killing by whole body bioluminescence imaging. To find the best treatment route and optimal dose of T cells in GC mice model, ICAM-1-CAR was injected via tail vein and intraperitoneal administration (IP) at two different doses, i.e., low dose (1 million CAR T) and high dose (10 million live T cells). Results: The ICAM-1 expression was higher in advanced stages (22.2% in stage II vs. 48.8% in stage III, p=0.002) and the patients with high ICAM-1 expression showed significantly poor survival (disease free survival, hazard ration [HR], 4.55, p<0.001; overall survival, HR, 3.89, p<0.001). The efficacy of ICAM-1-CAR T cells in vitro showed a strong correlation with the level of ICAM-1 expression in target cells, i.e., faster killing of GC with higher ICAM-1 expression. ICAM-1-CART facilitated regression of tumor in MKN-28 xenograft IP model. Additionally, the efficacy of ICAM-1-CAR was more prominent in mice treated with high dose of T cell and IP route. Compared to no treatment, ICAM-1CAR via IP led to tumor reduction that persisted for over 80 days and significantly improved survival without toxicity (p=0.049). Conclusion: ICAM-1 specific CAR T cells demonstrated significant therapeutic efficacy in vitro and in vivo against preclinical GC models. ICAM-1-CAR T cells may be developed into a promising treatment strategy for patients with ICAM-1 positive GC cancers. Citation Format: Minkyu Jung, Marjan Zaman, Ygindra Vedvyas, Xianglan Zhang, Jaclyn E. McCloskey, Yanping Yang, Irene M. Min, Raza Zamegar, Yoon Young Choi, Jae-Ho Cheong, Sung Hoon Noh, Sun Young Rha, Hyun Cheol Chung, Moonsoo M. Jin. Chimeric antigen receptor T cell therapy targeting ICAM-1 in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1442.

Augmentation of Nab-Paclitaxel Chemotherapy Response by Mechanistically Diverse Antiangiogenic Agents in Preclinical Gastric Cancer Models.

Gastric adenocarcinoma (GAC) remains the third most common cause of cancer-related deaths worldwide. Systemic chemotherapy is commonly recommended as a fundamental treatment for metastatic GAC; however, standard treatment has not been established yet. Angiogenesis plays a crucial role in the progression and metastasis of GAC. We evaluated therapeutic benefits of mechanistically diverse antiangiogenic agents in combination with nab-paclitaxel, a next-generation taxane, in preclinical models of GAC. Murine survival studies were performed in peritoneal dissemination models, whereas tumor growth studies were performed in subcutaneous GAC cell-derived or patient-derived xenografts. The mechanistic evaluation involved IHC and Immunoblot analysis in tumor samples. Nab-paclitaxel increased animal survival that was further improved by the addition of antiangiogenic agents ramucirumab (or its murine version DC101), cabozantinib and nintedanib. Nab-paclitaxel combination with nintedanib was most effective in improving animal survival, always greater than 300% over control. In cell-derived subcutaneous xenografts, nab-paclitaxel reduced tumor growth while all three antiangiogenic agents enhanced this effect, with nintedanib demonstrating the greatest inhibition. Furthermore, in GAC patient-derived xenografts the combination of nab-paclitaxel and nintedanib reduced tumor growth over single agents alone. Tumor tissue analysis revealed that ramucirumab and cabozantinib only reduced tumor vasculature, whereas nintedanib in addition significantly reduced tumor cell proliferation and increased apoptosis. Effects of nab-paclitaxel, a promising chemotherapeutic agent for GAC, can be enhanced by new-generation antiangiogenic agents, especially nintedanib. The data suggest that nab-paclitaxel combinations with multitargeted antiangiogenic agents carry promising potential for improving clinical GAC therapy. Mol Cancer Ther; 17(11); 2353-64. ©2018 AACR.

Abstract 5807: The dual c-Met/VEGFR2 inhibitor foretinib augments chemotherapy response in preclinical models of gastric cancer

Abstract BACKGROUND: Gastric adenocarcinoma (GAC) is the fourth most common malignant tumor in the world. Several growth factors and their receptors including c-Met and VEGFR, are overexpressed in GAC and thus provide a potentially effective therapeutic target. Foretinib is a novel small molecule inhibitor of the c-Met and VEGFR pathways. We evaluated the therapeutic efficacy of foretinib to enhance the antitumor response of nab-paclitaxel (NPT), a water-soluble albumin-bound formulation of paclitaxel, or oxaliplatin in preclinical models of GAC. METHODS: Tumor growth experiments were performed in subcutaneous xenografts in NOD/SCID mice using 5x106 MKN-45 cells. Animal survival study was performed as peritoneal dissemination model in NOD/SCID mice using 10x106 MKN-45 cells. The mechanistic evaluation involved immunohistochemical and Immunoblot analyses. RESULTS: In subcutaneous GAC xenografts, NPT and foretinib monotherapies demonstrated inhibition in tumor growth, while NPT plus foretinib combined showed additive effects. Net tumor growth in different therapy groups was 581.7 mm3 in controls, 397.9 mm3 after oxaliplatin, 229.9 mm3 after NPT, -82.6 mm3 (tumor regression) after foretinib, -74.1 mm3 after oxaliplatin+foretinib and -96.3 mm3 after NPT+foretinib. No significant change in body weight was observed for those mice treated with nab-paclitaxel, oxaliplatin or foretinib. In the GAC survival model, median animal survival compared to controls (23 days) remained unchanged after oxaliplatin therapy (24 days, p=ns) but increased after monotherapy with NPT (42 days, an 83% increase, p=0.0014) or foretinib (46 days, a 100% increase, p=0.0006). Importantly, a further increase in animal survival was observed in combination therapy groups: oxaliplatin+foretinib (55 days, a 139% increase, p=0.0006) and NPT+foretinib (76 days, 230% increase, p=0.0006). Effects of therapy on intratumoral proliferation and microvessel density corresponded with tumor growth inhibition data. In vitro studies demonstrated inhibition in the proliferation of GAC cells by both NPT and foretinib, with additive effects in combination. Immunoblot analysis revealed that foretinib pre-exposure blocked HGF-induced expression of phospho-c-Met. Furthermore, the foretinib treatment caused a decrease in phosphorylation of AKT, ERK, and PLC-γ in GAC cells, either alone or in combination with nab-paclitaxel. CONCLUSION: These findings suggest that the antitumor effect of chemotherapy can be significantly enhanced by the c-Met/VEGFR pathway inhibitor foretinib, which might lead to clinically relevant therapeutic combinations to increase survival of GAC patients. Citation Format: Niranjan Awasthi, Meghan Grojean, Sheena Monahan, Sazzad Hassan, Urs von Holzen, Margaret A. Schwarz, Roderich E. Schwarz. The dual c-Met/VEGFR2 inhibitor foretinib augments chemotherapy response in preclinical models of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5807.

Abstract 4633: MM-111, a bispecific HER2 and HER3 antibody, synergistically combines with trastuzumab and paclitaxel in preclinical models of gastric cancer.

Abstract Increasing evidence suggests amplification or overexpression of human epidermal growth factor receptor-2 (HER2), and HER3 levels are correlated to decreased survival in gastric cancers. Our previous studies established that MM-111, a novel bispecific antibody that specifically targets the HER2/HER3 heterodimer, blocks heregulin (HRG) binding to HER3 and corresponding downstream signaling pathways. In this study we used computational and experimental biology to assess the activity of MM-111 combination therapies in treating gastric cancer. First, we utilized a multi-scale computational network model of HER2-positive tumors that relates HER family signal transduction with cell growth to predict that MM-111 plus trastuzumab would synergistically inhibit tumor growth. The mechanism underlying this prediction is that the combinatorial blockade of HER2 and HER3 suppresses signal propagation through both the AKT and ERK cascades and this leads to synergistic cell growth arrest. Cell viability and signaling experiments in a gastric cancer model (NCI-N87) confirmed these predictions. In particular, MM-111 plus trastuzumab caused 25% greater cell growth inhibition than the additive effects of the individual treatments (compared to Bliss independence; p< 0.05). Next, we assessed the effect of exogenous HRG on this combination therapy. HRG reduced the activity of trastuzumab but did not affect MM-111’s activity, and it enhanced the synergy of this combination (50% greater than Bliss independence; p < 10−8). We then assessed the effect of adding paclitaxel to the trastuzumab plus MM-111 combination. Our key finding was that activation of HER3 by HRG greatly reduced paclitaxel's activity. MM-111, but not trastuzumab, restored cell sensitivity to paclitaxel in the presence of HRG. A synergistic effect (35% greater than Bliss independence; p < 10−8) was observed for the combination of MM-111, trastuzumab and paclitaxel. Similar combination effects were also achieved using a three-dimensional NCI-N87 spheroid assay: Inhibition of HER2, HER3, and the AKT and ERK pathways correlated with spheroid growth inhibition. Furthermore, HRG-overexpressing NCI-N87 (NCI-N87-HRG) xenografts were less sensitive to paclitaxel than the wild type xenografts. MM-111 restored the sensitivity to paclitaxel in these xenografts, even when given as a second line treatment regimen after tumor progression on the front-line regimen (trastuzumab plus 5-fluorouracil). Trastuzumab alone did not overcome the HRG-induced paclitaxel resistance, but its combination with MM-111 showed significantly greater activity than either drug alone. Taken together, our data suggest that combination of MM-111, trastuzumab and paclitaxel is worthy of investigation as a potential therapeutic strategy for the treatment of HER2-positive gastric cancer. Citation Format: Bo Zhang, Johanna Lahdenranta, Jinyan Du, Daniel Kirouac, Stephanie Nguyen, Ryan Overland, Violette Paragas, Arthur Kudla, Ulrik Nielsen, Charlotte McDonagh, Matthew Onsum. MM-111, a bispecific HER2 and HER3 antibody, synergistically combines with trastuzumab and paclitaxel in preclinical models of gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4633. doi:10.1158/1538-7445.AM2013-4633

Abstract 1052: Refametinib (BAY 86-9766): An allosteric inhibitor of MEK1/2, potently inhibits tumor growth in patient-derived xenograft models of gastric cancer.

Abstract Background: Gastric cancer (GC) is a prevalent and deadly disease. Because the Ras/Raf/MEK/ERK signaling cascade plays a critical role in signal transduction pathways that regulate growth factor response, angiogenesis, tumor cell proliferation, and apoptosis, blocking this signaling pathway could thus have therapeutic efficacy. This study aims to target this pathway in preclinical models of GC with refametinib (BAY 86-9766, RDEA119), an orally available, allosteric inhibitor of MEK1/2. Methods: Eight patient-derived GC xenograft models with different histotypes were treated orally with refametinib or vehicle and tumor growth was monitored using caliper measurements. Tissue sections of vehicle- or refametinib-treated xenografts were analyzed by immunohistochemistry for cell proliferation, apoptosis and microvessel area, using antibodies against p-Histone H3Ser10, cleaved PARP and CD31, respectively. Tumor lysates were analyzed by Western Blotting with antibodies for effects on marker proteins for proliferation, apoptosis and angiogenesis.Results: Refametinib demonstrated significant tumor growth inhibition ranging from 66-87% in eight GC models at a dose of 15 mg/kg/d. Refametinib inhibited cell proliferation by 2-4 fold as observed by reduced staining for p-Histone H3Ser10. Increased staining for caspase-cleaved p85 PARP by 1.5 to 9 fold was detected, indicating that refametinib induced apoptosis. Furthermore the compound significantly inhibited angiogenesis in 2 of 8 models by 3-fold compared to the vehicle-treated group as indicated by reduced CD31 staining. Reduced cell proliferation and induction of apoptosis were associated with decreased levels of p-ERK1/2, p-cdk2Thr14/Tyr15, p-p90RSKSer359/363, p-p70S6KThr421/Ser424, p-4EBP1Thr70, p-S6RSer235/236 and p-eIF4ESer209 and elevation of p-AktSer473, Bim and cleaved PARP. Conclusion: We show that single-agent refametinib has significant anti-tumor activity in patient-derived gastric cancer preclinical models. Our findings support further investigation of refametinib either in monotherapy or in combination with inhibitors of the PI3K-AKT-mTOR pathway in the treatment of GC. Citation Format: Huynh T. Hung, Richard Ong, Khee Chee Soo, Florian Puehler, Arne Scholz, Dominik Mumberg, Dieter Zopf, Karl Ziegelbauer. Refametinib (BAY 86-9766): An allosteric inhibitor of MEK1/2, potently inhibits tumor growth in patient-derived xenograft models of gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1052. doi:10.1158/1538-7445.AM2013-1052

Preclinical activity of MM-111, a bispecific ErbB2/ErbB3 antibody in previously treated ErbB2-positive gastric and gastroesophageal junction cancer.

48 Background: ErbB2 (HER2) overexpression has been reported in 7-34% of gastric cancers. ErbB3 (HER3) is the preferred dimerization partner of ErbB2, and ErbB2/ErbB3 heterodimer activation is implicated in the progression and metastasis of ErbB2+ tumors. Activation of ErbB3 signaling is a postulated resistance mechanism to current ErbB2-directed therapies and select chemotherapies. In line with this research, ErbB3 levels are associated with poor prognosis in gastric cancers. MM-111 is a bi-specific antibody that docks to ErbB2 and inhibits ErbB3 signaling in cells that overexpress ErbB2. In this study, MM-111 was evaluated in ErbB2+ gastric cancer by testing the activity of MM-111 in ErbB2+ pre-clinical models of gastric cancer, and by assessing the prevalence of potentially predictive biomarkers in a panel of archived gastric and gastroesophageal junction (GEJ) tumors. Methods: MM-111 was tested in ErbB2+ gastric cancer xenografts that were either untreated or after tumors ceased to respond to trastuzumab/5-FU. Xenografts were analyzed at multiple time points for the expression of ErbB-receptor family members and their downstream signaling by Luminex -assays. Preclinical data indicate that ErbB2, ErbB3, and heregulin are predictive biomarkers for MM-111. In order to determine the prevalence of our potentially predictive biomarkers in gastric and GEJ cancers, we obtained commercially archived tumor tissue and assayed the tissue for ErbB2 and ErbB3 expression levels using quantitative IHC, and measured heregulin transcript levels by RT-PCR. Results: MM-111 synergizes with various treatment regimens in the 2nd line treatment setting in ErbB2+ gastric cancer xenografts. In our models, the combination of MM-111, trastuzumab, and paclitaxel is particularly effective after tumors progressed on trastuzumab/5-FU. MM-111 inhibits the activity of the ErbB –signaling axis in these models. In addition, 23% of GEJ tumor samples and 20% of gastric samples were positive for potentially predictive biomarkers. Conclusions: ErbB2+xenograft tumors that stop responding to trastuzumab-based therapies benefit from MM-111–based regimens.

Real-time bioluminescence and tomographic imaging of gastric cancer in a novel orthotopic mouse model

Gastric cancer is the second leading cause of cancer mortality worldwide. Understanding the multistep process of carcinogenesis of gastric cancer is pivotal to develop novel therapeutic strategies. Molecular imaging in preclinical cancer models bridges the gap of laboratory-based experiment and clinical translation. To this end, the human gastric cancer cell line SGC-7901 was established to stably express luciferase and GFP by lentiviral transduction (SGC7901-Luc-GFP). Preclinical models were developed by orthotopic transplantation of SGC-7901-Luc-GFP into the sub-serosal layer of the stomach of immunocompromised mice. Tumor progression and therapeutic responses were dynamically tracked by bioluminescence imaging (BLI). Bioluminescence tomography (BLT) was used to monitor stereoscopic morphological and signal changes during tumor progression. Good correlation between cell number and bio-luminescence/fluorescence intensity was observed (R(2)=0.9983/r(2)=0.9974) in vitro. Tumor progression and therapeutic response could be successfully followed directly by BLI. Importantly, BLT provided a more accurate spatial location and tomographic quantification of the internal lesion. In conclusion, our novel bioluminescence-based preclinical gastric cancer models enable superior, noninvasive monitoring gastric cancer progression and their drug responses. The BLT technique in particular, may have great potential for future oncological studies.

Abstract B2: Regorafenib, an oral multi-kinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases, potently inhibits tumor growth and angiogenesis in patient-derived xenograft models of gastric cancer.

Abstract Background: Gastric cancer (GC) is a prevalent and deadly disease. Because the VEGFR and PDGFR signaling cascade plays a critical role in the development and progression of gastric cancer and VEGF expression is correlated with angiogenesis, tumor progression and poor prognosis in patients with GC, blocking these signaling pathways could thus have therapeutic efficacy. This study aims to investigate the potential of a combined therapeutic approach to target these pathways in preclinical models of GC with regorafenib an oral multi-kinase inhibitor targeting VEGFR1–3, PDGFR, FGFR, TIE-2, RET and KIT. Methods: Eight patient-derived GC xenograft models with different histotypes were treated orally with regorafenib or vehicle and tumor growth was monitored using caliper measurements. Tissue sections of vehicle- or regorafenib-treated xenografts were analyzed by immunohistochemistry for apoptosis, microvessel area, and cell proliferation using antibodies against cleaved PARP, CD31 and phospho-Histone3Ser10, respectively. Tumor lysates were analyzed by Western Blotting with respective antibodies for effects on marker proteins for proliferation, apoptosis and angiogenesis. Results: Regorafenib demonstrated significant tumor growth inhibition of 81%, 85% and 88% in the GC09-0109 model after oral administration of 5, 10 and 15 mg/kg/d, respectively. In addition potent tumor growth inhibition ranging from 72–96% was observed in seven other GC models at a dose of 10 mg/kg/d. Regorafenib significantly inhibited angiogenesis in all models by 4–12 fold compared to the vehicle-treated group, independent of their degree of vascularization, as indicated by reduced CD31 staining. Furthermore inhibition of cell proliferation by 2–4 fold was observed by reduced staining for p-histone 3Ser10. Increased staining for caspase-cleaved p85 PARP by 2 to 20 fold was detected, indicating that regorafenib induced apoptosis. Finally, regorafenib also induced tumor necrosis as detected by histological staining. Inhibition of angiogenesis, reduced cell proliferation and induction of apoptosis and necrosis was associated with decreased levels of p-ERK1/2, p-cdk2Thr14/Tyr15, p-VEGFR2Tyr951, p-p70S6KThr421/Ser424, p-S6RSer235/236 and p-eIF4ESer209 and elevation of cleaved caspase-3 and cleaved PARP. Conclusion: We show that regorafenib is an efficacious anti-tumor targeted agent in patient-derived gastric cancer preclinical models. Our findings support further investigation of regorafenib in the treatment of GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B2.

Abstract 4424: Antitumor activities of targeted therapeutic agents in preclinical models of gastric cancer

Abstract Gastric cancer (GC) is one of the most prevalent cancer types in Asia, and the 2nd most common cause of cancer deaths worldwide. Although considerable advances have been made with the use of improved surgical techniques and chemotherapy regimens, the prognosis for most GC patients remains poor. Therefore there is an urgent medical need for novel, effective targeted therapeutic agents for GC. One of the key drivers for a successful drug discovery program is utilization of predictive preclinical models that represent the patients of interest. Through internal efforts and collaborations we have developed subcutaneous (SC), orthotopic, syngeneic and patient-derived xenograft (PDX) models for target validation and pharmacological studies. Here we report the establishment of various cell line-derived mouse models of GC, and their validation with targeted therapeutic agents. In SC models of MKN-74, NCI-N87 and MKN-45, sunitinib malate significantly inhibited tumor growth. To establish orthotopic GC model, luciferase-expressing MKN-74 and NCI-N87 cells were first SC implanted in athymic nude mice. Four weeks after in vivo propagation, tumor fragments (1 mm3) were surgically implanted to the stomach wall in the greater curvature region. Primary tumor growth and spontaneous metastasis were monitored by bioluminescent imaging. Sunitinib malate treatment inhibited primary tumor growth but the effect on metastasis was inconclusive. Furthermore, PF-299804, an orally available irreversible pan-HER tyrosine kinase inhibitor, inhibited in vivo growth of NCI-N87 (with ERBB2 amplification), but not of AGS (without ERBB2 amplification). Both sunitinib malate and PF-299804 were well tolerated by the animals during the course of the studies. These results support anti-angiogenesis and as a valid approach for effective treatment of GC, and preclinically demonstrated the importance of patient selection for targeted agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4424. doi:10.1158/1538-7445.AM2011-4424