Colorectal Cancer Xenograft Model Research Articles

Karyopherin β1 deletion suppresses tumor growth and metastasis in colorectal cancer (CRC) by reducing MET expression

Colorectal cancer (CRC) has become the third most common type of cancer worldwide, and CRC liver metastasis (CRLM) is associated with poor survival rates. However, the molecular mechanisms driving this phenomenon remain unclear. Karyopherin β1 (KPNB1) is an adaptor protein that transports several proteins to the nuclear, and has been reported to play essential role in regulating many cancer-associated pathologies. Nevertheless, its role in CRC is unknown. The study was aimed to explore the role of KPNB1 as a pro-metastatic factor and to reveal the underlying mechanism. Here, the results indicated that KPNB1 expression was markedly increased in CRC samples. KPNB1 expression was gradually up-regulated with CRC development and was tightly correlated with poor prognosis in CRC patients. In vitro results demonstrated that KPNB1 decreasing markedly reduced CRC cell proliferation, migration and invasion, which was positively associated with the expression of MET proto-oncogene (MET). Further analysis revealed that KPNB1 decrease down-regulated the expression of epithelial-mesenchymal transition (EMT)-associated signals. In vivo experiments also demonstrated that KPNB1 knockdown evidently inhibited the tumor growth and metastasis in a CRC xenograft model. Importantly, we found that KPNB1 could interact with MET to modulate cell proliferation and metastasis in CRC. A subsequent mechanistic study illustrated that MET over-expression markedly eliminated KPNB1 silence-inhibited migration and invasion in CRC cells. In summary, KPNB1 deletion repressed the metastasis of CRC cells through interacting with MET, which could be served as a potential prognostic biomarker in CRC patients.

Identification of profilin 1 as the primary target for the anti-cancer activities of Furowanin A in colorectal cancer.

Furowanin A (Fur A) is a flavonoid compound isolated from medicinal plant Millettia pachycarpa Benth. This study aims to explore the effect of Fur A on Colorectal cancer (CRC) and its molecular mechanisms. Cell proliferative capacity of CRC cells was assessed by CCK-8 assay. Cell apoptosis and cell cycle distribution were detected by flow cytometry. Cell migration and invasion were detected by wound healing and Transwell assay, respectively. EMT markers, apoptosis and profilin 1(Pfn1) expression were detected by immunohistochemistry (IHC). The protein expression levels were examined by western blotting. i-TRAQ analyses were conducted to identify the differentially expressed genes in CRC cells. CRC xenograft model was also used to validate the in vivo anti-cancer activity of Fur A. Fur A exhibited anti-prolifertive, blocked cell cycle progression and promoted apoptotic cell death in CRC cells. Fur A suppressed the migration, invasion and epithelial-to-mesenchymal transition (EMT) in vitro, and tumor growth and pulmonary metastasis in vivo, without causing obvious toxicity. iTRAQ analysis identified Pfn1 as a gene up-regulated by Fur A. In xenograft tumor tissue, the expression of Pfn1 was also elevated by Fur A treatment. In clinical CRC samples, high expression of Pfn1 was correlated with lower stage and longer survival. Knockdown of Pfn1 significantly dampened the pro-apoptotic and anti-metastatic activities of Fur A in CRC cells. Ectopic Pfn1 expression augmented the anti-neoplastic activities of Fur A. Fur A exhibited anti-cancer activities in vitro and in vivo in CRC by up-regulating Pfn1.

Nitrogen Permease Regulator-Like-2 Exhibited Anti-Tumor Effects And Enhanced The Sensitivity Of Colorectal Cancer Cells To Oxaliplatin And 5-Fluorouracil.

BackgroundColorectal cancer (CRC) is one of the most common malignant tumors in the world. Our previous study revealed that nitrogen permease regulator-like-2 (NPRL2), a promising anti-tumor gene, was downregulated at both the blood and tissue levels in CRC patients compared with that in healthy individuals.PurposeThis study aims to explore the role of NPRL2 in CRC.MethodsHerein, we constructed NPRL2 overexpression lentivirus vectors and transfected them into HT29 cells. The transfected cells were inoculated subcutaneously into nude mice. Tumor growth, pathology, apoptosis, and the protein expression of caspase-3, caspase-7, Bax, Bcl-2, and phosphorylated protein kinase B (p-Akt) were evaluated. To further explore whether NPRL2 could reduce drug resistance of CRC cells against oxaliplatin (L-OHP) and 5-fluorouracil (5-FU), we constructed a tumor model using HT29 cells. The tumor model was treated with lentiviral particles assembled with vectors encoding NPRL2 and exposed to L-OHP and 5-FU. Tumor growth, pathology, apoptosis, and the protein expression of caspase-3, caspase-7, Bax, Bcl-2, p-Akt, P-glycoprotein (P-gp), and multidrug resistance protein 1 (MRP1) were evaluated.ResultsThe results indicated that in the in vivo CRC xenograft model, NPRL2 reduced the tumor volume and weight and enhanced apoptosis. Our results also confirmed that NPRL2 enhanced the sensitivity of CRC cells to L-OHP and 5-FU. Our studies further demonstrated that NPRL2 exerted anti-tumor and anti-drug resistance effects through the caspase-3, caspase-7, Bax, Bcl-2, Akt, P-gp, and MRP1 pathways.ConclusionOur present work demonstrated that NPRL2 exhibited anti-tumor effects and enhanced the sensitivities of CRC cells to L-OHP and 5-FU through the P-gp and MRP1 pathways.

A pretargeted multimodal approach for image-guided resection in a xenograft model of colorectal cancer

BackgroundImage-guided surgery may improve surgical outcome for colorectal cancer patients. Here, we evaluated the feasibility of a pretargeting strategy for multimodal imaging in colorectal cancer using an anti-carcinoembryonic antigen (CEA) x anti-histamine-succinyl-glycine (HSG) bispecific antibody (TF2) in conjunction with the dual-labeled diHSG peptide (RDC018), using both a fluorophore for near-infrared fluorescence imaging and a chelator for radiolabeling.MethodsNude mice with subcutaneous (s.c) CEA-expressing LS174T human colonic tumors and CEA-negative control tumors were injected with TF2. After 16 h, different doses of 111In-labeled IMP-288 (non-fluorescent) or its fluorescent derivative RDC018 were administered to compare biodistributions. MicroSPECT/CT and near-infrared fluorescence imaging were performed 2 and 24 h after injection. Next, the biodistribution of the dual-labeled humanized anti-CEA IgG antibody [111In]In-DTPA-hMN-14-IRDye800CW (direct targeting) was compared with the biodistribution of 111In-RDC018 in mice with TF2-pretargeted tumors, using fluorescence imaging and gamma counting. Lastly, mice with intraperitoneal LS174T tumors underwent near-infrared fluorescence image-guided resection combined with pre- and post-resection microSPECT/CT imaging.Results111In-RDC018 showed specific tumor targeting in pretargeted CEA-positive tumors (21.9 ± 4.5 and 10.0 ± 4.7% injected activity per gram (mean ± SD %IA/g), at 2 and 24 hours post-injection (p.i.), respectively) and a biodistribution similar to 111In-IMP288. Both fluorescence and microSPECT/CT images confirmed preferential tumor accumulation. At post mortem dissection, intraperitoneal tumors were successfully identified and removed using pretargeting with TF2 and 111In-RDC018.ConclusionA pretargeted approach for multimodal image-guided resection of colorectal cancer in a preclinical xenograft model is feasible, enables preoperative SPECT/CT, and might facilitate intraoperative fluorescence imaging.

Effects of metformin on tumor hypoxia and radiotherapy efficacy: a [18F]HX4 PET imaging study in colorectal cancer xenografts

BackgroundIn a colorectal cancer xenograft model, we investigated the therapeutic effect of metformin on tumor hypoxia with [18F]flortanidazole ([18F]HX4) small-animal positron emission tomography (μPET). We also assessed the additive effect of metformin on long-term radiotherapy outcome and we studied the potential of [18F]HX4 as a predictive and/or prognostic biomarker within this setup.MethodsColo205-bearing mice (n = 40) underwent a baseline [18F]HX4 hypoxia μPET/computed tomography (CT) scan. The next day, mice received 100 mg/kg metformin or saline intravenously (n = 20/group) and [18F]HX4 was administered intravenously 30 min later, whereupon a second μPET/CT scan was performed to assess changes in tumor hypoxia. Two days later, mice were further divided into four therapy groups (n = 10/group): control (1), metformin (2), radiotherapy (3), and metformin + radiotherapy, i.e., combination (4). Then, they received a second dose of metformin (groups 2 and 4) or saline (groups 1 and 3), followed by a single radiotherapy dose of 15 Gy (groups 3 and 4) or sham irradiation (groups 1 and 2) 30 min later. Tumor growth was followed three times a week by caliper measurements to assess the therapeutic outcome.Results[18F]HX4 uptake decreased in metformin-treated tumors with a mean intratumoral reduction in [18F]HX4 tumor-to-background ratio (TBR) from 2.53 ± 0.30 to 2.28 ± 0.26 (p = 0.04), as opposed to saline treatment (2.56 ± 0.39 to 3.08 ± 0.39; p = 0.2). The median tumor doubling time (TDT) was 6, 8, 41, and 43 days in the control, metformin, radiotherapy and combination group, respectively (log-rank p < 0.0001), but no metformin-specific therapy effects could be detected. Baseline [18F]HX4 TBR was a negative prognostic biomarker for TDT (hazard ratio, 2.39; p = 0.02).ConclusionsMetformin decreased [18F]HX4 uptake of Colo205-tumors, but had no additive effect on radiotherapy efficacy. Nevertheless, [18F]HX4 holds promise as a prognostic imaging biomarker.

Mesothelin-Targeted Thorium-227 Conjugate (MSLN-TTC): Preclinical Evaluation of a New Targeted Alpha Therapy for Mesothelin-Positive Cancers.

Targeted thorium-227 conjugates (TTC) represent a new class of molecules for targeted alpha therapy (TAT). Covalent attachment of a 3,2-HOPO chelator to an antibody enables specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. Because of the high energy and short penetration range, TAT efficiently induces double-strand DNA breaks (DSB) preferentially in the tumor cell with limited damage to the surrounding tissue. We present herein the preclinical evaluation of a mesothelin (MSLN)-targeted thorium-227 conjugate, BAY 2287411. MSLN is a GPI-anchored membrane glycoprotein overexpressed in mesothelioma, ovarian, pancreatic, lung, and breast cancers with limited expression in healthy tissue. The binding activity and radiostability of BAY 2287411 were confirmed bioanalytically. The mode-of-action and antitumor potency of BAY 2287411 were investigated in vitro and in vivo in cell line and patient-derived xenograft models of breast, colorectal, lung, ovarian, and pancreatic cancer. BAY 2287411 induced DSBs, apoptotic markers, and oxidative stress, leading to reduced cellular viability. Furthermore, upregulation of immunogenic cell death markers was observed. BAY 2287411 was well-tolerated and demonstrated significant antitumor efficacy when administered via single or multiple dosing regimens in vivo. In addition, significant survival benefit was observed in a disseminated lung cancer model. Biodistribution studies showed specific uptake and retention of BAY 2287411 in tumors and enabled the development of a mechanistic pharmacokinetic/pharmacodynamic model to describe the preclinical data. These promising preclinical results supported the transition of BAY 2287411 into a clinical phase I program in mesothelioma and ovarian cancer patients (NCT03507452).

Dietary methionine influences therapy in mouse cancer models and alters human metabolism.

SummaryNutrition exerts profound effects on health and dietary interventions are commonly used to treat diseases of metabolic etiology. Although cancer has a substantial metabolic component1, the principles that define whether nutrition may be used to influence tumour outcome are unclear2. Nevertheless, it is established that targeting metabolic pathways with pharmacological agents or radiation can sometimes lead to controlled therapeutic outcomes. In contrast, whether specific dietary interventions could influence the metabolic pathways that are targeted in standard cancer therapies is not known. We now show that dietary restriction of methionine (MR), an essential amino acid, and the reduction of which has anti-aging and anti-obesogenic properties, influences cancer outcome through controlled and reproducible changes to one-carbon metabolism. This pathway metabolizes methionine and further is the target of a host of cancer interventions involving chemotherapy and radiation. MR produced therapeutic responses in chemoresistant RAS-driven colorectal cancer patient derived xenografts (PDXs) and autochthonous KRASG12D+/−;TP53−/− -driven soft tissue sarcomas resistant to radiation. Metabolomics revealed the therapeutic mechanisms to occur through tumour cell autonomous effects on the flux through one-carbon metabolism that impacted redox and nucleotide metabolism, thus interacting with the antimetabolite or radiation intervention. Finally, in a controlled and tolerated feeding study in humans, MR resulted in similar effects on systemic metabolism as obtained in responsive mice. These findings provide evidence that a targeted dietary manipulation can affect specific tumour cell metabolism to mediate broad aspects of cancer outcome.

Abstract 2198: PBI-200: A novel, brain penetrant, next generation pan-TRK kinase inhibitor

Abstract Fusions of NTRK 1, 2 and3 genes encoding the TRK family of receptor tyrosine kinases (TrkA, B and C, respectively) have been reported in 1-3% of all human cancers including lung and breast cancers. NTRK gene fusions are also reported in lung and breast tumors metastatic to the brain as well as in primary brain tumors including glioblastoma (3%), astrocytoma (3%) and pediatric glioma (40%). These gene fusions result in constitutive TRK kinase activity and act as oncogenic drivers of disease. First generation TRK kinase inhibitors have demonstrated clinical proof of concept in patients with tumors bearing NTRK gene fusions. PBI-200 is a novel, selective pan-TRK kinase inhibitor. In biochemical assays it inhibits TrkA, B and C with IC50 values of 0.45, 2.2 and 1.9 nM, respectively. This molecule is highly selective against a panel of 122 other protein kinases tested with the exception of Ros1 (IC50= 31 nM). Importantly, PBI-200 retains potency against resistance mutations reported in patients receiving first generation TRK kinase inhibitors. Biochemical IC50 values are 3.4 nM for the TrkA G595R gatekeeper mutation and 10 nM for the TrkA G667C mutant. PBI-200 potently inhibits proliferation of human tumor cell lines expressing TRK fusions including the KM-12 colorectal cancer cell line (TPM3-NTRK1 fusion; EC50 value = 22 nM) and the MO-91 acute myeloid leukemia cell line (ETV6-NTRK3 fusion; EC50 value = 3.5 nM). It also potently inhibits proliferation of rat BaF3 cells expressing oncogenic NTRK fusions and their resistant variants. In a subcutaneous xenograft model of KM-12 colorectal cancer, PBI-200 induces tumor stasis when dosed intraperitoneally at 15 or 30 mg/kg twice daily (93 and 100% tumor growth inhibition, respectively). The activity of PBI-200 was comparable or superior to that seen with the first generation TRK kinase inhibitors used as comparators in this model. PBI-200 has oral bioavailability in both rat and mouse. Importantly, PBI-200 demonstrates excellent brain penetration in rodent species, with brain/plasma AUC ratios of 3.9 in the mouse and 3.2 in the rat. This is in contrast to the poor brain penetration observed with the first generation TRK kinase inhibitors, larotrectinib and entrectinib.PBI-200 is efficacious in an intracranial KM-12 xenograft model when dosed intraperitoneally or orally, significantly slowing tumor growth and extending survival. Based on the activity of PBI-200 against clinically-relevant resistance mutations and its excellent brain penetration, this molecule has potential to be a next-generation TRK kinase inhibitor especially for primary brain tumors or brain metastatic lesions that harbor a NTRK gene fusion. PBI-200 is currently undergoing further characterization in IND enabling studies. Citation Format: Anthony Regina, Aram Elagoz, Vincent Albert, Jonathan Boudreault, Hong Wang, Marc Ouellet, Nicolas Brunei-Latour, Edith Bellavance, Peter White, Stephane Ciblat, W. Robert Bishop, Kollol Pal. PBI-200: A novel, brain penetrant, next generation pan-TRK kinase inhibitor [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 2198.

Abstract 2491: Engineering and preclinical activity of MM-201, a best-in-class TRAIL receptor agonist

Abstract Early attempts at using TNF superfamily members for anticancer therapies, TNF and FAS, led to serious systemic toxicities. However, the discovery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) introduced an agonist capable of killing tumor cells via apoptosis without the side effects observed with TNF and FAS agonists. First generation TRAIL agonists included a recombinant version of human TRAIL (dulanermin), as well as multiple DR4 and DR5 agonist antibodies. Despite some isolated responders, the initial clinical results were poor. The first-generation TRAIL agonists were limited by poor pharmacokinetics (the half-life for dulanermin was between 30 and 60 minutes) or by poor agonist activity and the need for Fc-mediated cross linking. Here we present the development and evaluation of two second generation TRAIL agonists, MM-201a and MM-201b. Both versions are composed of an IgG1 Fc fused to a single chain TRAIL trimer (Fc-scTRAIL). Mutations within the TRAIL domains, selected from a random mutagenesis library, were introduced to improve stability, expression, and DR5 binding. MM-201a has 5 mutations in each monomeric unit (R130G/N228S/I247V/Y213W/S215D) and MM-201b has 3 mutations in each monomer (R130G/N228S/I247V). In a panel of 27 colorectal carcinoma and sarcoma cell lines, both versions of MM-201 were observed to be significantly more active than all comparators, including the TRAIL cytokine and both DR4 and DR5 antibodies. MM-201a had a level of activity similar to ABBV-621, a single chain TRAIL fused to the N-terminus of an IgG1 Fc that is currently the subject of a Phase 1 trial. However, MM-201b was significantly more active than both MM-201a and ABBV-621, with up to 11-fold lower IC50 across a panel of 12 CRC cell lines. MM-201b treatment reduced cell viability to less than 20% in 10 out 12 colorectal cancer cell lines and in 8 of these cell lines, this was achieved at concentrations less than 1 nM. MM-201 also induced complete cell death at 1 nM or less in 3 of 8 synovial sarcoma and chondrosarcoma cell lines tested. For example, MM-201b reduced the viability of the SW-982 synovial sarcoma cell line to 17% at a dose of 1.5 pM, which is nearly twice the reduction in viability from the same dose of ABBV-621. We next evaluated both versions of MM-201 in multiple colorectal cancer and sarcoma patient-derived xenograft (PDX) models. In the Ewing’s sarcoma PDX model TM01617, MM-201b treatment resulted in 90% tumor growth inhibition. In the same model, treatment with 10 mg/kg docetaxel resulted in 73% growth inhibition; however, in combination with MM-201a, the same dose resulted in a 100% complete response rate. Similar results were observed in the SK-UT1 uterine sarcoma xenograft. Based on this evidence, we believe that MM-201b is best in class and, when combined with an appropriate patient selection strategy, has significant potential for the treatment of sarcomas and colorectal cancer in patients. Citation Format: Andrew J. Sawyer, Sara Ghassemifar, Christina Wong, Jennifer Richards, Stephanie Grabow, James Suchy, Alexander Koshkaryev, Maja Razlog, Eric Tam, Daryl C. Drummond. Engineering and preclinical activity of MM-201, a best-in-class TRAIL receptor agonist [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 2491.

Abstract 2051: BI 905711, a novel CDH17-targeting TRAILR2 agonist, effectively triggers tumor cell apoptosis and tumor regressions selectively in CDH17-positive colorectal cancer models

Abstract The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has the capability to induce apoptosis in a wide variety of tumor cells and has emerged as an important therapeutic concept for cancer treatment. To date however, TRAILR2 agonistic antibodies, designed to trigger tumor cell apoptosis, have only had limited clinical success due either to lack of efficacy or liver toxicity. BI 905711, a tetravalent bispecific antibody targeting both TRAILR2 and CDH17, was designed to overcome the disadvantages of current TRAILR2 antibodies. CDH17-dependent clustering of TRAILR2 permits BI 905711 to selectively induce apoptosis in CDH17-expressing tumor cells. CDH17 was selected as the anchor target due to its restricted expression, in particular a lack of expression in normal liver tissue, thereby avoiding the clinical hepatotoxicity associated with TRAILR2 agonism. Here, we report the preclinical activity of BI 905711 using colorectal cancer (CRC)-derived cell lines and patient-derived xenograft (PDX) models. We demonstrated that BI 905711 effectively triggered apoptosis in a broad range of CDH17-positive CRC tumor cells in vitro. Furthermore, BI 905711 potently initiated the apoptosis cascade as evidenced by a strong post-treatment increase of both caspase-8 and caspase-3/7. Importantly, induction of extrinsic apoptosis signaling by BI 905711 was strictly CDH17-dependent, as further demonstrated using a pair of CDH17-positive and -negative clones of the CRC cell line GP2d. When comparing the CDH17-negative clone with the parental cell line, the absence of CDH17 translated into a more than 1000-fold drop in BI 905711-dependent efficacy. Moreover, BI 905711 demonstrated single-agent tumor regressions in the GP2d colorectal cancer xenograft model. The antitumor efficacy of BI 905711 was further investigated in vivo in different CRC PDX models, showing effective tumor growth inhibition in a q14d dosing schedule. In summary, we demonstrated that BI 905711 potently triggers the extrinsic apoptosis pathway specifically in CDH17-positive tumor cells, which translates into significant tumor growth inhibition in CRC patient-derived xenograft tumors. BI 905711 is a first-in-class molecule that shows superiority to existing TRAILR2 agonists, represents a targeted strategy for the treatment of CRC and additional CDH17-positive cancer types. Together with its potential for a favorable safety profile, these data support our plan for a phase I trial of BI 905711 in these patient populations. Citation Format: Juan Manuel Garcia-Martinez, Andreas Wernitznig, Joerg Rinnenthal, Maria Antonietta Impagnatiello, Frank Hilberg, Craig Giragossian, Norbert Kraut, Mark Pearson, Klaus-Peter Kuenkele. BI 905711, a novel CDH17-targeting TRAILR2 agonist, effectively triggers tumor cell apoptosis and tumor regressions selectively in CDH17-positive colorectal 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 2051.

Abstract 220: U3-1402, a novel HER3-targeting ADC, inhibits the tumor growth of colorectal cancer xenografts

Abstract Background: HER3 (human epidermal growth factor receptor 3) is a member of the HER family and overexpressed in a broad range of cancer types including colorectal cancer. HER3 targeted therapies such as anti-HER3 antibodies have been investigated in clinical trials; however, no HER3-targeting therapy has been approved to date. U3-1402 is a novel antibody drug conjugate (ADC) composed of a fully human anti-HER3 antibody (patritumab) and a novel topoisomerase I inhibitor (DXd) which is conjugated via an enzymatically cleavable peptide-linker with high drug antibody ratio (DAR: 7-8 to 1). Purpose: To investigate the antitumor efficacy of U3-1402 in HER3-expressing colorectal cancer xenograft models. Material and methods: The following cell lines were used in this study; WiDr, Colo320DM, and DiFi with KRAS wild type, DLD-1, HCT15, HCT116, LoVo, SW480, and SW620 harboring KRAS mutation. The sensitivities of 9 colorectal cancer cell lines to DXd were evaluated by in vitro cell killing assay. Cells were treated with DXd at different concentrations (0.5pM to 500nM) over 144 hours, and the 50% inhibitory concentration (IC50) values were determined. Cell surface HER3 antigen was quantified by flow cytometry. In vivo screening assay and tumor growth inhibition assay were performed in tumor xenograft models exhibiting a range of HER3 expression levels. In addition, the efficacy of U3-1402 was compared to that of parental antibody patritumab, CPT-11, control-ADC, and saline in SW620 tumor xenograft model harboring KARS mutation. All results were expressed as mean ± standard deviation (SD) and statistical significance was analyzed using ANOVA with Dunnet multiple comparison. Results: In in vitro study, the range of IC50 value of DXd for each cell line was 2.16 ± 0.11 nM to 16.04 ± 0.10 nM. Most of the cell lines were sensitive to DXd. In in vivo screening assay, the maximum regression from U3-1402 was observed in the tumor xenograft model of both DiFi (high HER3 expression; KRAS wild type) and SW620 (high HER3 expression; KRAS mutation). In in vivo tumor growth inhibition assay, the antitumor efficacy was dependent on the HER3 expression level. Notably, SW620 tumor growth was significantly suppressed with U3-1402 compared with that of saline group (p &amp;lt; 0.001) and had improved potency compared with CPT-11. Of note, no significant difference in tumor growth inhibition was observed amongst the treatments with the same dose of patritumab or control-ADC and saline. Tumor regression was only observed in U3-1402 treated group, and some of which achieved complete regression. Conclusions: The antitumor efficacy of U3-1402 in xenograft models was dependent on HER3 expression and did not show a clear relationship to KRAS mutation status. These results support further investigation of development strategies for U3-1402 in patients with HER3-expressing colorectal cancer. Citation Format: Shigehiro Koganemaru, Yasutoshi Kuboki, Yoshikatsu Koga, Takashi Kojima, Mayumi Yamauchi, Naoyuki Maeda, Takashi Kagari, Kenji Hirotani, Masahiro Yasunaga, Yasuhiro Matsumura, Toshihiko Doi. U3-1402, a novel HER3-targeting ADC, inhibits the tumor growth of colorectal cancer xenografts [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 220.

Abstract 2821: Gut commensal bacteria modulate functions of tumor-associated neutrophils in human colorectal cancer

Abstract Tumor infiltration by immune cells critically impacts on clinical outcome of human colorectal cancer (CRC). While high densities of CD8+ T cells and T-helper type 1 cells are associated with prolonged patient survival, the role of tumor-associated neutrophils (TANs) is debated. CRC arise in an environment heavily populated by microrganisms. Upon CRC oncogenesis, gut commensal bacteria translocate into the submucosa where they directly interact with residing immune cells. Neutrophils represent a front-line arm of the immune system in the response to bacteria. However, little is known about their interaction with commensal bacteria within CRC tissues. We investigated the interplay between neutrophils and commensal bacterial species present within the CRC microenvironment. Using an orthotopic CRC xenograft model, we found that commensal bacteria stimulate CRC cells to produce neutrophil recruiting chemokines. We then compared in vitro chemokine induction capacity of Fusobacterium nucleatum (Fn) and Bacteroides fragilis (Bf), two most abundant bacterial species of CRC microenvironment. Fninduced significantly higher levels of IL-8 by CRC cells than Bf, thus more effectively promoting neutrophil recruitment. Accordingly, in human CRC samples, abundance of Fn, but not Bf, significantly correlated with high infiltration by CD66b+ cells. Functional studies indicate that neutrophils cultured in the presence of Fn, but not Bf, lose their ability to enhance proliferation and cytokine release by CD8+ T cells undergoing antigenic stimulation. Moreover, neutrophils exposed to Fn, but not Bf, stimulate release of IL-6 by tumor-associated stromal cells, leading to enhanced tumor cell proliferation. Our data cumulatively suggest that distinct bacterial components of the human gut flora might differentially modulate functions of CRC infiltrating neutrophils, thus ultimately influencing their prognostic significance. Note: This abstract was not presented at the meeting. Citation Format: Valeria Governa, Eleonora Cremonesi, Diego Calabrese, Valentina Mele, Emanuele Trella, Raoul A. Droeser, Martin Bolli, Serenella Eppenberger-Castori, Salvatore Piscuoglio, Charlotte K. y. Ng, Jesus Francisco Glaus Garzon, Lubor Borsig, Klaus-Peter Janssen, Luigi M. Terracciano, Giulio Cesare Spagnoli, Dimitri Christoforidis, Pietro Edoardo Majno-Hurst, Giandomenica Iezzi. Gut commensal bacteria modulate functions of tumor-associated neutrophils in human colorectal 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 2821.

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

Lipophilic Modification of an Anti‐Cancer Stem Cell Agent Improves Pharmacokinetic and Anti‐Cancer Properties

Current anti‐cancer agents target bulk tumor cells but do not eradicate the rare, quiescent, cancer stem cells (CSCs). CSCs are being increasingly recognized as the reason for cancer relapse. Targeting CSCs has become a major area of interest in the field of anti‐cancer drug discovery and several clinical trials focused on CSC targeting are in progress. Previously, we identified a CSC selective agent G2.2 (Fig. 1), which inhibited CSC spheroidal growth with a median IC50 (±1SEM) s of 193 μM±58 μM. G2.2 was found to exhibit robust anti‐CSC properties in colorectal cancer xenograft models in vivo. Mechanistically, G2.2 is a mimetic of heparan sulfate hexasaccharide (HS06) and is perhaps the only non‐saccharide heparan sulfate mimetic identified to date to exhibit considerable anti‐cancer promise. Preliminary pharmacokinetic (PK) studies showed that G2.2 exhibited a half‐life of nearly 4 hours following IV administration and 6% bioavailability following oral administration in rats.To improve upon G2.2's PK properties, we hypothesized lipid‐modification of G2.2 would enhance penetration across GI barrier while also simultaneously enhancing its potency through better targeting of plasma‐membrane growth factor receptors – the presumed targets of G2.2 and HS06. Several lipid‐modified agents were synthesized. These modifications resulted in improvement of PK properties (bioavailability by greater than 4‐fold). Further, the analogs also showed up to ~ 53‐fold (NCI‐H508 spheroids) enhanced inhibition potency against CSCs in in‐vitro spheroid cell‐based assays. In fact, these lipid‐modified G2.2 analogs inhibited several (&gt;10) colorectal cancer spheroids with median IC50 values (±1SEM) ranging from 8 μM±3μM to 68 μM±14μM. Considering that G2.2 is a mimetic of heparan sulfate, these results present a simple avenue to enhance drug‐like properties of such highly charged agents. The results also suggest that the lipid‐modified analogs may also serve as probes of molecular processes governing the CSC phenotype.Support or Funding InformationThis work was funded by the NHLBI grant 1P01HL107152 given to Umesh R. Desai.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BET Inhibitors Potentiate Chemotherapy and Killing of SPOP-Mutant Colon Cancer Cells via Induction of DR5

Bromodomain and extraterminal domain (BET) family proteins such as BRD4 are epigenetic readers that control expression of a number of oncogenic proteins. Targeting this family of proteins has recently emerged as a promising anticancer approach. BET inhibitors (BETi), either alone or in combination with other anticancer agents, have exhibited efficacy in a variety of tumors. However, the molecular mechanisms underlying differential response to BETi are not well understood. In this study, we report that death receptor 5 (DR5), a key component of the extrinsic apoptotic pathway, is markedly induced in response to BRD4 depletion and BETi treatment in colorectal cancer cells. Induction of DR5, following BET inhibition, was mediated by endoplasmic reticulum stress and CHOP-dependent transcriptional activation. Enhanced DR5 induction was necessary for the chemosensitization and apoptotic effects of BETi and was responsible for increased BETi sensitivity in colorectal cancer cells containing a mutation in speckle-type POZ protein (SPOP), a subunit of BRD4 E3 ubiquitin ligase. In a colorectal cancer xenograft model, BETi combined with chemotherapy suppressed the tumor growth in a DR5-dependent manner and potently inhibited patient-derived xenograft tumor growth with enhanced DR5 induction and apoptosis. These findings suggest that BETi alone or in combination with chemotherapy is effective against colorectal cancer due to enhanced DR5 induction and apoptosis. DR5 induction may also serve as a useful marker for designing personalized treatment and improved colorectal cancer combination therapies.Significance: These findings reveal how BET inhibition sensitizes chemotherapy and kills a subset of colon cancer cells with specific genetic alterations and may provide a new molecular marker for improving colon cancer therapies.

Activity of RX-3117, an oral antimetabolite nucleoside, in subjects with advanced urothelial cancer: Preliminary results of a phase IIa study.

455 Background: RX-3117 is an oral small molecule antimetabolite that is activated by uridine cytidine kinase 2 (UCK2) which is predominantly expressed in cancer cells. RX-3117 has shown efficacy in xenograft models of gemcitabine resistant pancreatic, bladder and colorectal cancer. Preliminary data from an analysis of a Phase 2a clinical study of RX3117 in advanced urothelial cancer is described. Methods: In the Phase 2a study designed to evaluate safety, tolerability and efficacy, subjects were treated with oral RX-3117 (700 mg) once-daily for 5 consecutive days on and 2 days off for 3 of 4 weeks or all 4 weeks in a 28-day cycle. Eligible subjects (aged ≥ 18 years) had relapsed/refractory metastatic urothelial cancer, ECOG PS of 0 to 1, normal organ function (hepatic, renal and hematology) with no limit on the number of prior therapies. The primary Phase 2a endpoints are progression free survival (PFS) and/or objective clinical response with secondary endpoints of safety, TTP, DOR and ORR. Results: As of October 5, 2018; 33 subjects were treated (23 males and 10 females, median age 67.5 years); 29 subjects were evaluable having completed more than 1 cycle of therapy or discontinued due to a related adverse event. Twenty subjects had received 3 or more prior therapies; 30 received gemcitabine/cisplatin and 25 received a checkpoint inhibitor. The most common related adverse events were anemia (G1-2%, G2-3%, G3-3%), fatigue (G1-6%, G2-3%), neutropenia (G2-2%, G3-5%, G4-2%), diarrhea (G1-4%, G2-2%), and thrombocytopenia (G2-2%, G3-3%, G4-1%). One subject had a complete response after 4 cycles of therapy and continues therapy beyond 10 cycles; 5 subjects had PFS ranging from 133 to 315 days. Conclusions: RX-3117 appears to be safe and well-tolerated in chemotherapy and immunotherapy refractory advanced urothelial cancer with acceptable toxicities. Preliminary results show anti-tumor activity in heavily pre-treated patients. Clinical trial information: NCT02030067.

Salinomycin: Anti-tumor activity in a pre-clinical colorectal cancer model.

ObjectivesSalinomycin is a polyether antibiotic with selective activity against human cancer stem cells. The impact of salinomycin on patient-derived primary human colorectal cancer cells has not been investigated so far. Thus, here we aimed to investigate the activity of salinomycin against tumor initiating cells isolated from patients with colorectal cancer.MethodsPrimary tumor-initiating cells (TIC) isolated from human patients with colorectal liver metastases or from human primary colon carcinoma were exposed to salinomycin and compared to treatment with 5-FU and oxaliplatin. TICs were injected subcutaneously into NOD/SCID mice to induce a patient-derived mouse xenograft model of colorectal cancer. Animals were treated either with salinomycin, FOLFOX regimen, or salinomycin and FOLFOX. Human colorectal cancer cells were used to delineate an underlying molecular mechanism of salinomycin in this tumor entity.ResultsApplying TICs isolated from human patients with colorectal liver metastases or from human primary colon carcinoma, we demonstrated that salinomycin exerts increased antiproliferative activity compared to 5-fluorouracil and oxaliplatin treatment. Consistently, salinomycin alone or in combination with FOLFOX exerts superior antitumor activity compared to FOLFOX therapy in a patient-derived mouse xenograft model of colorectal cancer. Salinomycin induces apoptosis of human colorectal cancer cells, accompanied by accumulation of dysfunctional mitochondria and reactive oxygen species. These effects are associated with expressional down-regulation of superoxide dismutase-1 (SOD1) in response to salinomycin treatment.ConclusionCollectively, the results of this pre-clinical study indicate that salinomycin alone or in combination with 5-fluorouracil and oxaliplatin exerts increased antitumoral activity compared to common chemotherapy.

Efficacy of a Covalent ERK1/2 Inhibitor, CC-90003, in KRAS-Mutant Cancer Models Reveals Novel Mechanisms of Response and Resistance.

As a critical signaling node, ERK1/2 are attractive drug targets, particularly in tumors driven by activation of the MAPK pathway. Utility of targeting the MAPK pathway has been demonstrated by clinical responses to inhibitors of MEK1/2 or RAF kinases in some mutant BRAF-activated malignancies. Unlike tumors with mutations in BRAF, those with mutations in KRAS (>30% of all cancers and >90% of certain cancer types) are generally not responsive to inhibitors of MEK1/2 or RAF. Here, a covalent ERK1/2 inhibitor, CC-90003, was characterized and shown to be active in preclinical models of KRAS-mutant tumors. A unique occupancy assay was used to understand the mechanism of resistance in a KRAS-mutant patient-derived xenograft (PDX) model of colorectal cancer. Finally, combination of CC-90003 with docetaxel achieved full tumor regression and prevented tumor regrowth after cessation of treatment in a PDX model of lung cancer. This effect corresponded to changes in a stemness gene network, revealing a potential effect on tumor stem cell reprograming. IMPLICATIONS: Here, a covalent ERK1/2 inhibitor (CC-90003) is demonstrated to have preclinical efficacy in models of KRAS-mutant tumors, which present a therapeutic challenge for currently available therapies.

Abstract A161: Targeting gastrointestinal tumors with constant region engineered anti-glycan antibodies

Abstract Post-translational modifications, for instance protein and lipid glycosylation, are attractive targets for therapeutic antibody (mAb) development. The altered tumor glyco-code drives oncogenic features such as the ability to proliferate, metastasize as well evade immune detection. Through immunizations with colorectal cancer cell membrane extracts we have generated a panel of anti-glycan mAbs with potential for application as cancer therapeutics. These mAbs selectively target Lewis or sialylated Lewis glycans on glycoproteins and/or glycolipids, binding to a large percentage of colorectal, pancreatic and gastric tumor tissues on tumor microarrays and induce a significant tumor volume reduction combined with a survival benefit in metastatic colorectal cancer xenograft models. Underlying these potent antitumor responses is the mAbs’ ability to induce direct tumor cell killing in the absence of complement or immune effector cells. In vitro, this direct cytotoxicity is characterized by mAb-induced cellular aggregation, pore formation, release of alarmins (ATP and high mobility group box 1 protein (HMGB1)), and maturation of immature dendritic cells, thereby constituting a form of inflammatory cell death (ICD). This mode of cell killing is linked to mAb cooperative binding on repeating antigen, a characteristic mostly associated with the murine mIgG3 isotype, thus human hIgG1 formats do not exhibit this form of direcT-cell killing. We have identified the residues required for the cooperative binding behaviour, through mAb constant region (CH2/CH3) screening, and have engineered improved (‘i’) hIgG1 variants, containing these selected residues. In silico immunogenicity prediction (IEDB) suggests limited immunogenicity, which requires further validation. Functional characterization of three improved anti-glycan hIgG1 mAbs in in vitro cell-based and Biacore assays demonstrates significant direct cancer cell killing, pore formation as well as increased functional glycan affinity. Classical immune effector functions (ADCC and CDC) were maintained or improved. Importantly, these improved hIgG1 variants now show significant tumor volume reduction in vivo in a mouse colorectal xenograft model.The multifaceted killing activity of our hIgG1 anti-glycan mAbs, has the potential to synergize with checkpoint blockade, thus holding tremendous therapeutic promise for the treatment of gastrointestinal tumors. Additionally, the activity of other therapeutic mAbs could be further enhanced with our Fc-engineering strategy for introducing cooperative binding. Citation Format: Mireille Vankemmelbeke, Thomas Kirk, Jia X. Chua, Richard McIntosh, Lindy G. Durrant. Targeting gastrointestinal tumors with constant region engineered anti-glycan antibodies [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A161.

Development of colloidal rare-earth arsenites as arsenic trioxide nanoparticle prodrugs (ATONP) for chemotherapy on a patient-derived xenograft model of colorectal cancer

Rare earth arsenite colloids, acting as arsenic trioxide nanoparticle prodrugs, effectively arrested cancer growth in a patient-derived colorectal xenograft model.