Colorectal Xenografts Research Articles

Platinum-Triggered Bond-Cleavage of Pentynoyl Amide and N-Propargyl Handles for Drug-Activation.

The ability to create ways to control drug activation at specific tissues while sparing healthy tissues remains a major challenge. The administration of exogenous target-specific triggers offers the potential for traceless release of active drugs on tumor sites from antibody–drug conjugates (ADCs) and caged prodrugs. We have developed a metal-mediated bond-cleavage reaction that uses platinum complexes [K2PtCl4 or Cisplatin (CisPt)] for drug activation. Key to the success of the reaction is a water-promoted activation process that triggers the reactivity of the platinum complexes. Under these conditions, the decaging of pentynoyl tertiary amides and N-propargyls occurs rapidly in aqueous systems. In cells, the protected analogues of cytotoxic drugs 5-fluorouracil (5-FU) and monomethyl auristatin E (MMAE) are partially activated by nontoxic amounts of platinum salts. Additionally, a noninternalizing ADC built with a pentynoyl traceless linker that features a tertiary amide protected MMAE was also decaged in the presence of platinum salts for extracellular drug release in cancer cells. Finally, CisPt-mediated prodrug activation of a propargyl derivative of 5-FU was shown in a colorectal zebrafish xenograft model that led to significant reductions in tumor size. Overall, our results reveal a new metal-based cleavable reaction that expands the application of platinum complexes beyond those in catalysis and cancer therapy.

Intratumoral exposure levels of pentaglutamated pemetrexed following treatment with LEAF-1401 and pemetrexed.

3524 Background: The activity of pemetrexed is highly dependent on the intracellular enzyme folypolyglutamate synthase (FPGS) which adds glutamates to pemetrexed and yields very potent pemetrexed polyglutamates. Pemetrexed pentaglutamate (tetraglutamated pemetrexed) is 80-fold more potent than pemetrexed in inhibiting thymidylate synthase. Yet it is a poor drug candidate because it cannot readily cross the negatively charged cell membrane due to its own negative charge. We are developing LEAF-1401, a novel nanoliposomal encapsulation of gamma L-pentaglutamated pemetrexed. Because liposomes can readily be taken up by tumor cells, for its anti-tumor effect, LEAF-1401 can directly deliver pentaglutamated pemetrexed into tumor cells, bypassing the need for transmembrane folate carriers and FPGS which are both downregulated in resistant tumors. Methods: To measure drug levels in tumor, blood and various tissues (biodistribution), in vivo testing of LEAF-1401 and pemetrexed was conducted in a CT-26 murine colorectal carcinoma xenograft model. Animals were treated with a single dose of either LEAF-1401 (80mg/kg; equivalent to 32 mg/kg pemetrexed) or pemetrexed (118mg/kg). Tumor growth inhibition and clinical assessments were conducted. Animals were sacrificed: 5 mice per timepoint in each group and tumor, blood, liver, spleen and other tissues were harvested. Pentaglutamated pemetrexed levels were quantitatively analyzed by LC/MS/MS. Results: Compared to pemetrexed, LEAF-1401 treatment resulted in a 19-fold increase in exposure levels of pentaglutamated pemetrexed in the tumor and significant tumor growth inhibition. Plasma levels of pentaglutamated pemetrexed were high with LEAF-1401, but undetectable with pemetrexed. Like other liposomes, LEAF-1401 also resulted in accumulation of pentaglutamated pemetrexed in the liver and spleen (See Table below). Treatment appeared to be generally well tolerated. Conclusions: LEAF-1401, given at approximately a quarter of the equivalent pemetrexed dose, resulted in a 19-fold increase in pentaglutamate pemetrexed in tumor tissue compared to regular pemetrexed. LEAF-1401 represents a promising new class of novel nanoliposomal antifolates, that enhance the intratumoral delivery of potent polyglutamate antifolates, and improve antitumor activity while retaining an acceptable safety profile. [Table: see text]

A Novel GUCY2C-CD3 T-Cell Engaging Bispecific Construct (PF-07062119) for the Treatment of Gastrointestinal Cancers.

Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy. PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3ε transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti-PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque. We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ε bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell-mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRAS- and BRAF-mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti-PD-1/PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile. These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies.

A reformulation of pLSA for uncertainty estimation and hypothesis testing in bio-imaging.

MotivationProbabilistic latent semantic analysis (pLSA) is commonly applied to describe mass spectra (MS) images. However, the method does not provide certain outputs necessary for the quantitative scientific interpretation of data. In particular, it lacks assessment of statistical uncertainty and the ability to perform hypothesis testing. We show how linear Poisson modelling advances pLSA, giving covariances on model parameters and supporting testing for the presence/absence of MS signal components. As an example, this is useful for the identification of pathology in MALDI biological samples. We also show potential wider applicability, beyond MS, using magnetic resonance imaging (MRI) data from colorectal xenograft models.ResultsSimulations and MALDI spectra of a stroke-damaged rat brain show MS signals from pathological tissue can be quantified. MRI diffusion data of control and radiotherapy-treated tumours further show high sensitivity hypothesis testing for treatment effects. Successful and degrees-of-freedom are computed, allowing null-hypothesis thresholding at high levels of confidence.Availability and implementationOpen-source image analysis software available from TINA Vision, www.tina-vision.net.Supplementary information Supplementary data are available at Bioinformatics online.

Plasticity in Motion: Shape-Shifting Lgr5-Cells Initiate Colorectal Cancer Metastasis.

In this issue of Cell Stem Cell, Fumagalli etal. (2020) employ intravital microscopy of colorectal cancer organoid xenografts to investigate the cell of origin of metastases. While tumor-initiating cells are Lgr5+, most disseminated cancer cells are Lgr5- and seed liver metastases in which Lgr5+ cells then appear, showing that bidirectional plasticity of phenotypic states drives metastasis.

Attenuation of the Hypoxia Inducible Factor Pathway after Oncolytic Adenovirus Infection Coincides with Decreased Vessel Perfusion.

The interplay between oncolytic virus infection and tumour hypoxia is particularly unexplored in vivo, although hypoxia is present in virtually all solid carcinomas. In this study, oncolytic adenovirus infection foci were found within pimonidazole-reactive, oxygen-poor areas in a colorectal xenograft tumour, where the expression of VEGF, a target gene of the hypoxia-inducible factor (HIF), was attenuated. We hypothesised that adenovirus infection interferes with the HIF-signalling axis in the hypoxic tumour niche, possibly modifying the local vascular supply. In vitro, enadenotucirev (EnAd), adenovirus 11p and adenovirus 5 decreased the protein expression of HIF-1α only during the late phase of the viral life cycle by transcriptional down-regulation and not post-translational regulation. The decreasing HIF levels resulted in the down-regulation of angiogenic factors such as VEGF, coinciding with reduced endothelial tube formation but also increased T-cell activation in conditioned media transfer experiments. Using intravital microscopy, a decreased perfused vessel volume was observed in infected tumour nodules upon systemic delivery of EnAd, encoding the oxygen-independent fluorescent reporter UnaG to a tumour xenograft grown under an abdominal window chamber. We conclude that the attenuation of the HIF pathway upon adenoviral infection may contribute to anti-vascular and immunostimulatory effects in the periphery of established infection foci in vivo.

FcRn overexpression in human cancer drives albumin recycling and cell growth; a mechanistic basis for exploitation in targeted albumin-drug designs

Albumin accumulation in tumours could reflect a role of albumin in transport of endogenous nutrient cargos required for cellular growth and not just a suggested source of amino acids; a role driven by albumin engagement with its cognate cellular recycling neonatal Fc receptor. We investigate the hypothesis that albumin cellular recruitment is increased by higher human FcRn (hFcRn) expression in human cancer tissue that provides the mechanistic basis for exploitation in albumin-based drug designs engineered to optimise this process. Eight out of ten different human cancer tissue types screened for hFcRn expression by immunohistochemistry (310 samples) exhibited significantly higher hFcRn expression compared to healthy tissues. Accelerated tumour growth over 28 days in mice inoculated with hFcRn-expressing HT-29 human colorectal cancer cell xenografts, compared to CRISPR/Cas9 hFcRn-knockout HT-29, suggests a hFcRn-mediated tumour growth effect. Direct correlation between hFcRn expression and albumin recycling supports hFcRn-mediated diversion of albumin from lysosomal degradation. Two-fold increase in accumulation of fluorescent labelled high-binding hFcRn albumin, compared to wild type albumin, in luciferase MDA-MB-231-Luc-D3H2LN breast cancer xenografts was shown. This work identifies overexpression of hFcRn in several human cancer types with mechanistic data suggesting hFcRn-driven albumin recruitment for increased cellular growth that has the potential to be exploited with high hFcRn-binding albumin variants for targeted therapies.

Temozolomide and Pazopanib Combined with FOLFOX Regressed a Primary Colorectal Cancer in a Patient-derived Orthotopic Xenograft Mouse Model

Purpose: The goal of the present study was to determine the efficacy of temozolomide (TEM) and pazopanib (PAZ) combined with FOLFOX (oxaliplatin, leucovorin and 5-fluorouracil) on a colorectal cancer patient-derived orthotopic xenograft (PDOX) mouse model. Materials and Methods: A colorectal cancer tumor from a patient previously established in non-transgenic nude mice was implanted subcutaneously in transgenic green fluorescence protein (GFP)-expressing nude mice in order to label the tumor stromal cells with GFP. Then labeled tumors were orthotopically implanted into the cecum of nude mice. Mice were randomized into four groups: Group 1, untreated control; group 2, TEM + PAZ; group 3, FOLFOX; group 4, TEM + PAZ plus FOLFOX. Tumor width, length, and mouse body weight were measured weekly. The Fluor Vivo imaging System was used to image the GFP-lableled tumor stromal cells in vivo. H&E staining and immunohistochemical staining were used for histological analysis. Results: All three treatments inhibited tumor growth as compared to the untreated control group. The combination of TEM + PAZ + FOLFOX regressed tumor growth significantly more effectively than TEM + PAZ or FOLFOX. Only the combination of TEM + PAZ + FOLFOX group caused a decrease in body weight. PAZ suppressed lymph vessels density in the colorectal cancer PDOX mouse model suggesting inhibition of lymphangiogenesis. Conclusion: Our results suggest that the combination of TEM + PAZ + FOLFOX has clinical potential for colorectal cancer patient.

Reovirus Enhances Cytotoxicity of Natural Killer Cells Against Colorectal Cancer via TLR3 Pathway

Colorectal cancer (CRC) is currently the fourth most common cause of cancer-associated deaths. Treatment with cetuximab, a chimeric immunoglobulin G1 (IgG1) monoclonal antibody targeting epidermal growth factor receptor (EGFR), has shown limited efficacy as a CRC monotherapy. In addition, natural killer (NK) cell function is known to be severely attenuated in cancer patients. Here, w e describe a viable combination strategy to enhance NK cell cytotoxicity while also improving the effects of cetuxim ab -mediated antibody-dependent cell-mediated cytotoxicity (ADCC). Reovirus can directly activate NK cells in vitro, and NK cells can transport reovirus to tumors, where it preferentially replicates and lyses tumor cells. We first confirmed that exposure of NK cells to reovirus enhanced their cytotoxicity in a dose-dependent manner in vitro. We then investigated whether reovirus-activated NK cells exposed to cetuximab-bound CRC cells exhibited greater anti-tumor efficacy than either monotherapy. Co-culture of three separate CRC cell lines with reovirus-activated NK cells indicated that NK cytotoxicity was significantly higher in combination with cetuximab, regardless of KRAS mutation status or EGFR expression level. We also found that reovirus activation of NK cells, in conjunction with cetuximab, resulted in significantly stronger anti-tumor effects in colorectal tumor xenograft models.Finally, T oll-like receptor 3 ( TLR3) knockdown in NK cells, combined with chemical blockade of TLR3/dsRNA complex, and inhibition of the TLR3 downstream signaling pathway, demonstrated that activation of NK cells by reovirus is dependent on TLR3 and its downstream signaling pathway. This study demonstrates that combination treatment of reovirus-activated NK cells with cetuximab synergistically enhances their anti-tumor cytotoxicity, suggesting a strong candidate strategy for clinical treatment of CRC. Funding Statement: This work was supported by the National Natural Science Foundation of China (No.81860542), the Guizhou Provincial Natural Science Foundation [grant no. (2019)5663], the Program for Top Scientific and Technological Talents in Guizhou Province [grant no. KY (2018)049];Non-Profit Central Research Institute Fund of Chinese Academy of Medical Sciences(No. 2018PT31048、2019PT310013). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were approved by the Ethics Committee of Guizhou Medical University (Ref: 2000803).

Oxidative stress-driven DR5 upregulation restores TRAIL/Apo2L sensitivity induced by iron oxide nanoparticles in colorectal cancer

There exists an emergency clinical demand to overcome TRAIL/Apo2L (tumor necrosis factor-related apoptosis-inducing ligand) resistance, which is a major obstacle attributed to insufficient level or mutation of TRAIL receptors. Here, we developed an iron oxide cluster-based nanoplatform for both sensitization and MR image-guided evaluation to improve TRAIL/Apo2L efficacy in colorectal cancer, which has an inadequate response to TRAIL/Apo2L or chemotherapy. Specifically, NanoTRAIL (TRAIL/Apo2L-iron oxide nanoparticles) generated ROS (reactive oxygen species)-triggered JNK (c-Jun N-terminal kinase) activation and induced subsequent autophagy-assisted DR5 upregulation, resulting in a significant enhanced antitumor efficacy of TRAIL/Apo2L, which confirmed in both TRAIL-resistant HT-29, intermediately resistant SW-480 and sensitive HCT-116 cells. Furthermore, in a subcutaneous colorectal cancer mouse model, the in vivo tumor retention of NanoTRAIL can be demonstrated by MR T2 weighted contrast imaging, and NanoTRAIL significantly suppressed tumor growth and prolonged the survival time without observable adverse effects compared with control and TRAIL/Apo2L monotherapy. Importantly, in the study of colorectal cancer patient-derived xenograft models, we found that the NanoTRAIL treatment could significantly improve the survival outcome with consistent ROS-dependent autophagy-assisted DR5 upregulation and tumor apoptosis. Our results describe a transformative design that can be applied clinically to sensitize Apo2L/TRAIL-resistant patients using FDA-approved iron oxide nanoparticles.

An N-Acetylgalactosamino Dendron-Clearing Agent for High-Therapeutic-Index DOTA-Hapten Pretargeted Radioimmunotherapy.

Clearing agents (CAs) can rapidly remove nonlocalized targeting biomolecules from circulation for hepatic catabolism, thereby enhancing the therapeutic index (TI), especially for blood (marrow), of the subsequently administered radioisotope in any multistep pretargeting strategy. Herein we describe the synthesis and in vivo evaluation of a fully synthetic glycodendrimer-based CA for DOTA-based pretargeted radioimmunotherapy (DOTA-PRIT). The novel dendron-CA consists of a nonradioactive yttrium-DOTA-Bn molecule attached via a linker to a glycodendron displaying 16 terminal α-thio-N-acetylgalactosamine (α-SGalNAc) units (CCA α-16-DOTA-Y3+; molecular weight: 9059 Da). Pretargeting [177Lu]LuDOTA-Bn with CCA α-16-DOTA-Y3+ to GPA33-expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [177Lu]LuDOTA-Bn for blood, tumor, liver, spleen, and kidneys of 11.7, 468, 9.97, 5.49, and 13.3 cGy/MBq, respectively. Tumor-to-normal tissues absorbed-dose ratios (i.e., TIs) ranged from 40 (e.g., for blood and kidney) to about 550 for stomach.

Abstract B007: OKI-179 is a novel, oral, class I specific histone deacetylase inhibitor in phase 1 clinical trials

Abstract Multiple histone deacetylase (HDAC) inhibitors are approved for use as single agents in hematological cancers, including T-cell lymphomas and multiple myeloma. However, these compounds have failed to address unmet needs in other hematological malignancies and solid tumors, due in part to the promiscuous inhibitory properties of pan inhibitors, a lack of suitable therapeutic indices for combination therapy, and, in some cases, a non-oral route of administration. Of the approved agents, romidepsin is the most potent and selective, but is limited by its need for IV infusion and therapeutic index. With these challenges in mind, our team designed OKI-179, a novel, oral pro-drug analog of largazole, a compound reminiscent of the romidepsin-depsipeptide class of natural products. OKI-179 demonstrates potent inhibition of the Class 1 HDACs, HDAC 1,2,3 (IC50 = 1.2, 2.4, 2.0 nM, respectively), with no significant inhibition of Class IIa HDACs. It shows potent antiproliferative and apoptotic effects against a wide range of cell lines, activity in xenograft cancer models, and good oral PK properties in mouse, rat and dog models. OKI-179 inhibits the growth in mice of colorectal (HCT-116) and triple negative breast (MDA-MB-231) xenograft models potently and dose-dependently as a single agent. OKI-179 potently and dose-dependently causes the upregulation of MHC Class 1 in B16F10 murine melanoma cells; patients with low MHC presentation respond poorly to checkpoint inhibitors. Importantly, the combination of OKI-179 and a murine anti-PD1 agent demonstrates significant synergistic tumor growth inhibition in the syngeneic CT26 murine CRC model and in a genetic model of DLBCL. OKI-179 is currently being investigated in a first-in-human phase 1 dose-escalation trial, with the goals of determining safety, MTD, RP2D, PK, PD, and preliminary efficacy signals. In the first dosing cohorts, the drug was well-tolerated, achieved good exposure following oral administration, and preliminary signs of on-target PD effects were observed. An update of the clinical experience will be presented and discussed, along with translational data that will guide subsequent clinical development. Citation Format: Jennifer R Diamond, Jodi A Kagihara, Xuedong Liu, Gilad Gordon, Amy M Heim, James Winkler, John A DeMattei, Anthony D Piscopio, S. Gail Eckhardt. OKI-179 is a novel, oral, class I specific histone deacetylase inhibitor in phase 1 clinical trials [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B007. doi:10.1158/1535-7163.TARG-19-B007

Development of Novel Silyl Cyanocinnamic Acid Derivatives as Metabolic Plasticity Inhibitors for Cancer Treatment

Novel silyl cyanocinnamic acid derivatives have been synthesized and evaluated as potential anticancer agents. In vitro studies reveal that lead derivatives 2a and 2b have enhanced cancer cell proliferation inhibition properties when compared to the parent monocarboxylate transporter (MCT) inhibitor cyano-hydroxycinnamic acid (CHC). Further, candidate compounds exhibit several-fold more potent MCT1 inhibition properties as determined by lactate-uptake studies, and these studies are supported by MCT homology modeling and computational inhibitor-docking studies. In vitro effects on glycolysis and mitochondrial metabolism also illustrate that the lead derivatives 2a and 2b lead to significant effects on both metabolic pathways. In vivo systemic toxicity and efficacy studies in colorectal cancer cell WiDr tumor xenograft demonstrate that candidate compounds are well tolerated and exhibit good single agent anticancer efficacy properties.

Promising Therapeutic Efficacy of GC1118, an Anti-EGFR Antibody, against KRAS Mutation-Driven Colorectal Cancer Patient-Derived Xenografts.

Epidermal growth factor receptor (EGFR)-targeted monoclonal antibodies, including cetuximab and panitumumab, are used to treat metastatic colorectal cancer (mCRC). However, this treatment is only effective for a small subset of mCRC patients positive for the wild-type KRAS GTPase. GC1118 is a novel, fully humanized anti-EGFR IgG1 antibody that displays potent inhibitory effects on high-affinity EGFR ligand-induced signaling and enhanced antibody-mediated cytotoxicity. In this study, using 51 CRC patient-derived xenografts (PDXs), we showed that KRAS mutants expressed remarkably elevated autocrine levels of high-affinity EGFR ligands compared with wild-type KRAS. In three KRAS-mutant CRCPDXs, GC1118 was more effective than cetuximab, whereas the two agents demonstrated comparable efficacy against three wild-type KRAS PDXs. Persistent phosphatidylinositol-3-kinase (PI3K)/AKT signaling was thought to underlie resistance to GC1118. In support of these findings, a preliminary improved anti-cancer response was observed in a CRC PDX harboring mutated KRAS with intrinsically high AKT activity using GC1118 combined with the dual PI3K/mammalian target of rapamycin (mTOR)/AKT inhibitor BEZ-235, without observed toxicity. Taken together, the superior antitumor efficacy of GC1118 alone or in combination with PI3K/mTOR/AKT inhibitors shows great therapeutic potential for the treatment of KRAS-mutant mCRC with elevated ratios of high- to low-affinity EGFR ligands and PI3K-AKT pathway activation.

Therapeutic Dose Response of Acoustic Cluster Therapy in Combination With Irinotecan for the Treatment of Human Colon Cancer in Mice.

Introduction: Acoustic Cluster Therapy (ACT) comprises coadministration of a formulation containing microbubble-microdroplet clusters (PS101) together with a regular medicinal drug and local ultrasound (US) insonation of the targeted pathological tissue. PS101 is confined to the vascular compartment and when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase shift to produce bubbles with a median diameter of 22 µm. Low frequency, low mechanical index US is then applied to drive oscillations of the deposited ACT bubbles to induce biomechanical effects that locally enhance extravasation, distribution, and uptake of the coadministered drug, significantly increasing its therapeutic efficacy. Methods: The therapeutic efficacy of ACT with irinotecan (60 mg/kg i.p.) was investigated using three treatment sessions given on day 0, 7, and 14 on subcutaneous human colorectal adenocarcinoma xenografts in mice. Treatment was performed with three back-to-back PS101+US administrations per session with PS101 doses ranging from 0.40–2.00 ml PS101/kg body weight (n = 8–15). To induce the phase shift, 45 s of US at 8 MHz at an MI of 0.30 was applied using a diagnostic US system; low frequency exposure consisted of 1 or 5 min at 500 kHz with an MI of 0.20. Results: ACT with irinotecan induced a strong, dose dependent increase in the therapeutic effect (R2 = 0.95). When compared to irinotecan alone, at the highest dose investigated, combination treatment induced a reduction in average normalized tumour volume from 14.6 (irinotecan), to 5.4 (ACT with irinotecan, p = 0.002) on day 27. Median survival increased from 34 days (irinotecan) to 54 (ACT with irinotecan, p = 0.002). Additionally, ACT with irinotecan induced an increase in the fraction of complete responders; from 7% to 26%. There was no significant difference in the therapeutic efficacy whether the low frequency US lasted 1 or 5 min. Furthermore, there was no significant difference between the enhancement observed in the efficacy of ACT with irinotecan when PS101+US was administered before or after irinotecan. An increase in early dropouts was observed at higher PS101 doses. Both mean tumour volume (on day 27) and median survival indicate that the PS101 dose response was linear in the range investigated.

Efficacy of a Bispecific Antibody Co-Targeting VEGFA and Ang-2 in Combination with Chemotherapy in a Chemoresistant Colorectal Carcinoma Xenograft Model.

Vascular endothelial growth factor (VEGF) inhibition by the addition of bevacizumab to the chemotherapy regimen of metastatic colorectal cancer leads to an improved outcome. However, anti-angiogenic tumor therapy targeting a single factor may be limited by complementary mechanisms. Angiopoietin-2 (Ang-2, ANGPT2) is another important factor that cooperates with VEGF to drive tumor angiogenesis. It was shown that high Ang-2 levels are associated with a poor clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy. Therefore, combined inhibition of VEGF and Ang-2 was supposed to improve anti-angiogenic therapy. Here, we evaluated the efficacy of a bispecific antibody (CrossMab) co-targeting VEGF and Ang-2 in combination with chemotherapy in a chemoresistant colorectal carcinoma model. Antitumor activity was evaluated in athymic nude mice bearing subcutaneous DLD1 xenograft tumors and treated with anti-VEGF (B20), anti-Ang-2 (LC06) and anti-VEGF/Ang-2 (CrossMab) antibodies. Chemotherapy consisted of 5-FU and irinotecan. Resected tumors were analyzed immunohistochemically. First, an impact of targeting each single factor but also a clear advantage of co-targeting both factors could be demonstrated. Accordingly, tumor tissue showed strong staining for VEGF and Ang-2. Chemotherapy alone was less effective. Efficient tumor growth inhibition could be achieved by treatment with anti-VEGF/chemotherapy, single CrossMab and CrossMab/chemotherapy, which resulted in 3 out of 10, 6 out of 10 and 10 out of 10 complete responses, respectively, during seven weeks. Complete retarded tumors were characterized by massive intratumoral necrosis surrounded by layers of vital tumor cells and connective tissue with CD31-positive vessels at the periphery. In some cases, a distinct feature known as vessel co-option could be observed. In conclusion, the data from this model clearly support the strategy of co-targeting VEGF and Ang-2 and further demonstrate the beneficial impact of co-treatment with chemotherapy. The clear superiority of the CrossMab-containing regimen compared to clinical standard anti-VEGF/chemotherapy warrants further analyses in other models.

Discovery and Optimization of 2-Arylquinazolin-4-ones into a Potent and Selective Tankyrase Inhibitor Modulating Wnt Pathway Activity.

Tankyrases 1 and 2 (TNKS1/2) are promising pharmacological targets that recently gained interest for anticancer therapy in Wnt pathway dependent tumors. 2-Aryl-quinazolinones were identified and optimized into potent tankyrase inhibitors through SAR exploration around the quinazolinone core and the 4'-position of the phenyl residue. These efforts were supported by analysis of TNKS X-ray and WaterMap structures and resulted in compound 5k, a potent, selective tankyrase inhibitor with favorable pharmacokinetic properties. The X-ray structure of 5k in complex with TNKS1 was solved and confirmed the design hypothesis. Modulation of Wnt pathway activity was demonstrated with this compound in a colorectal xenograft model in vivo.

Mechanically and chemically defined hydrogel matrices for patient-derived colorectal tumor organoid culture

Patient-derived tumor organoids offer potentially useful models of cancer tissue physiology. Yet, conventional organoid cultures utilize generic matrices that are difficult to tailor for various unique tumor microenvironments. Here, we employ synthetic, enzymatically crosslinked hydrogels to define mechanical and biochemical properties hypothesized to be relevant for maintaining these organoids. We show that a single extracellular matrix component, gelatin, suffices to support colorectal cancer patient-derived xenograft (CRC-PDX) organoid survival, and that high matrix stiffness synergizes with hypoxia to increase organoid growth and metabolism in a majority of CRC-PDX lines tested. Moreover, we demonstrate that defined gelatin-based hydrogels support CRC-PDX tumor growth in vivo and organoid sensitivity to various CRC therapeutic drugs in vitro in a largely comparable fashion to a conventional reconstituted basement membrane matrix. Based on our findings, we propose that enzymatically crosslinked hydrogels potentially provide a platform for designing mechanically and biochemically defined matrices for various types of patient-derived tumor organoids.

Salinomycin exerts anti-colorectal cancer activity by targeting the β-catenin/T-cell factor complex.

Background and PurposeSalinomycin is a well‐known inhibitor of human cancer stem cells (CSCs). However, the molecular mechanism(s) by which salinomycin targets colorectal CSCs is poorly understood. Here, we have investigated underlying antitumour mechanisms of salinomycin in colorectal cancer cells and three tumour models.Experimental ApproachThe inhibitory effect of salinomycin on the Wnt/β‐catenin pathway was analysed with the SuperTopFlash reporter system. The mRNA expression of Wnt target genes was evaluated with real‐time PCR. Effects of salinomycin on β‐catenin/TCF4E interaction were examined using co‐immunoprecipitation and an in vitro GST pull‐down assay. Cell proliferation was determined by BrdU incorporation and soft agar colony formation assay. The stemness of the cells was assessed by sphere formation assay. Antitumour effects of salinomycin on colorectal cancers was evaluated with colorectal CSC xenografts, APCmin/+ transgenic mice, and patient‐derived colorectal tumour xenografts.Key ResultsSalinomycin blocked β‐catenin/TCF4E complex formation in colorectal cancer cells and in an in vitro GST pull‐down assay, thus decreasing expression of Wnt target genes. Salinomycin also suppressed the transcriptional activity mediated by β‐catenin/LEF1 or β‐catenin/TCF4E complex and exhibited an inhibitory effect on the sphere formation, proliferation, and anchorage‐independent growth of colorectal cancer cells. In colorectal tumour xenografts and APCmin/+ transgenic mice, administration of salinomycin significantly reduced tumour growth and the expression of CSC‐related Wnt target genes including LGR5.Conclusions and ImplicationsOur study suggested that salinomycin could suppress the growth of colorectal cancer by disrupting the β‐catenin/TCF complex and thus may be a promising agent for colorectal cancer treatment.

Abstract 353: A novel molecule with profound tumor killing activity

Abstract LY3343544: A novel MET antibody drug conjugate that shows profound pre-clinical in vivo anti-tumor activities, irrespective of MET pathway dependence MET is over-expressed in many types of human tumors. Due to the heterogeneity of human tumors, MET antibodies or small molecule inhibitors have benefited only small subsets of patients with tumors driven by signaling through the c-Met pathway. The patient selection strategies to identify those tumors with MET activation dependence are helpful in predicting sensitivity to many of these inhibitors. It was reported previously that Lilly’s MET antibody, emibetuzumab, showed clinical activity in selective NSCLC patients with high MET IHC staining (90% to 100% 3+ positive) when it was combined with erlotinib in Phase II clinical Trials. In searching for a better treatment for patients carrying the MET overexpression tumors regardless other co-existing mutations, we developed LY3343544, a novel antibody drug conjugate (ADC) molecule that consists of emibetuzumab conjugated with the potent microtubule inhibitor MMAE using a unique lysine conjugation approach. Upon binding to MET, LY3343544 is internalized via receptor-mediated endocytosis. LY3343544 maintains the similar binding and internalization activities to the cell surface MET as compared to emibetuzumab in the competitive cell binding assay and the internalization assay. We reported here that LY3343544 showed profound anti-tumor activity in a preclinical mouse models, and overcome intrinsic resistance mechanisms including KRAS, BRAF, PI3K and TP53 mutations. LY3343544 kills tumor cells expressing a wide range of MET levels on the cell surface and is capable of killing a variety of MET-overexpressing tumor cells including pancreatic, cholanglocarcinoma, colorectal, NSCLC, gastric, head and neck tumor cells in vitro. In contrast, LY3343544 does not kill human normal endothelial cells and normal epithelial cells, no activity on human peripheral blood mononuclear cells with or without activation as well as in cell-based assays. Moreover, LY3343544 is more stable in rodent PK studies than typical inter chain Cys VC-MMAE conjugates and showed tumor regressions in colorectal, NSCLC, gastric and pancreatic mouse xenograft models. Furthermore, LY3343544 shows profound tumor regression in >50% of PDAC PDX models (n=40): 20% complete response (CR); 22.5% partial response (PR); and 17.5% stable disease (SD); overall disease control rate (DCR) is 60%. In addition, LY3343544 shows tumor growth inhibition in cholangiocarcinoma PDX model that is resistant to emibetuzumab. In summary, LY3343544 is highly potent in killing a variety of tumor cells in cell-based killing assays. It demonstrated good stability in vivo and profound anti-tumor efficacy in multiple mouse xenograft models and patient-derived xenograft models thus is a promising agent to treat many types of cancers. Citation Format: Ling Liu, Aaron D. Wrobleski, Yin Yin, Wei Zeng, Xianming Chen, David J. Stokell, Sheng-bin Peng, Amita Datta-Mannan, Gregory P. Donoho, Philip W. Iversen, Philip Hipskind, Yiqing Feng. A novel molecule with profound tumor killing activity [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 353.