Tumor-derived Xenograft Research Articles

Anlotinib Combined With Chemoradiotherapy Exhibits Significant Therapeutic Efficacy in Esophageal Squamous Cell Carcinoma.

Objective: The aim of this study was to evaluate the safety and efficacy of anlotinib combined with chemoradiotherapy for treating esophageal squamous cell carcinoma (ESCC) using patient-derived xenografts (PDXs).Methods: PDX-bearing mice were randomly divided into five groups, as follows: control group receiving normal saline, the group receiving radiotherapy, the group receiving cisplatin combined with radiotherapy, the group receiving anlotinib combined with radiotherapy, and the group receiving anlotinib, and cisplatin combined with radiotherapy. Tumor volumes and body weights were measured three times weekly for 2 weeks. The PDXs were initially assessed by comparing the histology of the original patient tumor tissues with that of the corresponding serially passaged xenografts by hematoxylin and eosin (H&E) and P63 staining. Then, expression of Bax, c-PARP, PCNA, and CD31 was detected using immunohistochemistry, and apoptosis was detected by a TUNEL assay. Cytokines released into plasma were analyzed using protein chip technology. Finally, two case studies of ESCC patients were presented to further verify the results observed in the PDX models.Results: The pathological characteristics of the serially passaged patient tumor-derived xenografts established in our study were in line with those of the original ESCC patient samples. The group receiving anlotinib and cisplatin plus radiotherapy exhibited the strongest antitumor response among the groups. Moreover, the ideal anticancer effects of anlotinib combined with chemoradiotherapy observed in clinical patients were consistent with the results observed in the PDX models, and no serious side effects were observed during treatment.Conclusions: Combination therapy with anlotinib and chemoradiotherapy may be an effective regimen for the treatment of advanced ESCC.

Dietary Flavonoids Targeting Triple Negative Breast Cancer

Abstract Objectives Breast cancer remains the leading cause of malignancy-related death in females and continues to increase in prevalence worldwide. The lack of therapies for triple-negative breast cancer (TNBC) continues to be a challenge, owing to its highly metastatic and aggressive nature. Dietary flavonoids, found in fruits and vegetables, are attracting great interest in the prevention and treatment of TNBC due to their anti-carcinogenic, anti-oxidative, and anti-inflammatory properties. The goal of our project was to evaluate the structure-activity relationship and the mechanism by which flavonoids affect TNBC tumor growth and metastasis. Methods We investigated the effects of structurally related flavonoids on the migratory nature of human MDA-MB 231 TNBC cells by using a wound healing migration assay. To model the effects of flavonoids in tumor heterogeneity in vivo, 3-dimensional culture organoids from TNBC derived xenograft tumors were established. Small interfering RNA (siRNA) transfection of apigenin target heterogeneous nuclear ribonucleoproteins A2 (hnRNPA2), an oncogene overexpressed in cancer cells that leads to abnormal mRNA splicing, were employed to investigate how hnRNPA2 effects alternative mRNA splicing activity. Results We observed that the natural flavonoids apigenin and kaempferol inhibited migration in a dose-response manner. Conversely, the presence of a glucoside and/or the lack of double bonds within the flavonoid structure as in the case of apigenin-7-glucoside and flavanones exhibited no significant anti-metastatic effects. Analysis of hnRNPA2 knockdown revealed key insights into the interaction of apigenin with hnRNPA2 to regulate migratory behaviors. Conclusions These novel insights showcase dietary flavonoids as practical functional foods that can benefit clinical applications in TNBC cancer prevention and treatment of tumor metastases and growth. Funding Sources Through grants from United States Department of Agriculture, National Science Foundation, and MSU awarded to Dr. Andrea I. Doseff, NIH-T32 Plant Biotechnology for Health and Sustainability Fellowship to Meenakshi Sudhakaran, and the REPID Program funded by National Institute of Health and directed by Dr. Elahé Crockett.

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Abstract B50: Tissue factor-targeting CAR-NK cells for immunotherapy of triple-negative breast cancer

Abstract The objective of the study is to develop tissue factor (TF)-targeted chimeric antigen receptor-natural killer (TF-CAR-NK) cell immunotherapy for the treatment of triple-negative breast cancer (TNBC). TNBC represents nearly 15% of globally diagnosed breast cancer, which is also the most frequently diagnosed cancer in women in the United States. Due to the lack of validated therapeutic target molecules, TNBC is one of the most difficult-to-treat malignancies, and, in most cases, is considered an incurable malignancy. CAR-expressing NK and T-cell therapy represents a novel cancer immunotherapy approach. To address the unmet issue, we first identified that TF is a novel, common, yet selective membrane-bound surface protein receptor and oncotarget for TNBC on the TNBC cancer cells, tumor neovascular endothelial cells and cancer stem cells. It is worth noting that under physiologic conditions, TF is not expressed on normal peripheral lymphocytes such as NK, B, T cells and monocytes, nor on the cells (quiescent vascular endothelial cells) forming the inner layer of normal vasculature, but is solely restricted on some cells (such as pericytes and smooth muscle cells) that are located outside the blood circulation and inner layer of blood vessel walls. Then we developed and tested a novel TF-targeting CAR-NK cell line (NK92MI, a human NK line derived from NK92 after being transfected for endogenous production of interleukin-2) expressing factor VII light chain, natural ligand for TF, as TF-targeting domain followed by CD28-4-1BB-CD3zeta with or without a hinge region of human IgG1 as TF-CAR1 dimer or monomer for CAR-NK immunotherapy of TNBC. Our preclinical results demonstrate that both the TF-CAR1 dimer and monomer-NK cells can kill TNBC cells in vitro and are effective and safe for the treatment of TNBC in mouse models of TNBC cell line-derived and patient’s tumor-derived xenografts (CDX and PDX). Thus, this study established the proof of concept of targeting TF for CAR-NK immunotherapy of TNBC and may warrant further investigation for TNBC patients. Accumulating evidence suggested that TF is selectively expressed in pathologic angiogenesis-dependent as well as macrophage-associated human diseases. Pathologic angiogenesis, the formation of neovasculature, is involved in many clinically significant human diseases, notably cancer (including solid cancer, acute leukemia and sarcoma), age-related macular degeneration, endometriosis and rheumatoid arthritis, whereas macrophage is involved in the progression of a variety of human diseases, such as atherosclerosis and viral infections (HIV and Ebola). If successful, TF-CAR immunotherapy may have potential to treat a broad range of angiogenesis-dependent or macrophage-associated human diseases. Acknowledgments: This work was supported by the Dr. Ralph and Marian Falk Medical Research Trust Awards Programs and partly by an NCI grant (R21CA216697). Z.H. is the inventor of PCT/U.S. Pending Patents on “Tissue Factor-Targeting CAR-NK and CAR-T Cell Therapy.” Citation Format: Zhiwei Hu. Tissue factor-targeting CAR-NK cells for immunotherapy of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B50.

Tissue factor as a new target for CAR-NK cell immunotherapy of triple-negative breast cancer

Triple-negative breast cancer (TNBC), representing ~15% of globally diagnosed breast cancer, is typically an incurable malignancy due to the lack of targetable surface targets for development of effective therapy. To address the unmet need for TNBC treatment, we recently determined that tissue factor (TF) is a useful surface target in 50–85% of patients with TNBC and developed a second-generation TF-targeting antibody-like immunoconjugate (called L-ICON) for preclinical treatment of TNBC. Using the chimeric antigen receptor (CAR) approach, here we develop and test TF-targeting CAR-engineered natural killer (TF-CAR-NK) cells that co-express CD16, the Fc receptor (FcγIII) to mediate antibody-dependent cellular toxicity (ADCC), for a preclinical assessment of immunotherapy of TNBC using TF-CAR-NK cell as single agent therapy and in combination with L-ICON. Our preclinical results demonstrate that TF-CAR-NK cells alone could kill TNBC cells and its efficacy was enhanced with L-ICON ADCC in vitro. Moreover, TF-CAR-NK cells were effective in vivo for the treatment of TNBC in cell line- and patient’s tumor-derived xenograft mouse models. Thus, this study established the proof of concept of targeting TF as a new target in CAR-NK immunotherapy for effective treatment of TNBC and may warrant further preclinical study and potentially future investigation in TNBC patients.

A systematical comparison of anti-angiogenesis and anti-cancer efficacy of ramucirumab, apatinib, regorafenib and cabozantinib in zebrafish model

AimsGastric cancer (GC) is one of the most common malignant tumors in the world. Anti-angiogenic therapy is a useful strategy for the treatment of advanced GC. This study was aimed to systemically compare the anti-angiogenesis, anti-cancer efficacy, as well as the safety of four known anti-angiogenic drugs, namely ramucirumab, apatinib, regorafenib and cabozantinib. Main methodsAnti-angiogenic effect was evaluated for the intersegmental vessels (ISVs) and subintestinal veins (SIVs) formation in the Tg (fli-1: EGFP) zebrafish embryos. Anti-cancer efficacy was tested for the in vivo cell proliferation in cell line derived tumor xenograft (CDX) model based on Tg (fli-1: EGFP) zebrafish embryos. Key findingsAll four drugs exhibited anti-angiogenic abilities and tumor inhibition effects in fli-1: EGFP transgenic zebrafish. Using zebrafish xenografted model, we found that effectiveness of ramucirumab in anti-GC-proliferation is better than apatinib, regorafenib and cabozantinib. The combination of anti-angiogenic drugs and cisplatin showed no significant benefit in tumors. Meanwhile, toxicity assay showed that all tested anti-angiogenic drugs could cause cardiovascular-related side effects. The therapeutic index (LD50/ED50) of cabozantinib is higher than apatinib and regorafenib, suggesting a potential as an anti-GC drug. SignificanceThe comparison of GC-related anti-angiogenic drugs was first reported. It was found that cabozantinib had a potential as an anti-GC drug. Zebrafish model was an ideal animal model for the research of anti-angiogenic behaviors.

Abstract C061: BT8009, a Bicycle Toxin Conjugate targeting Nectin-4, shows target selectivity, and efficacy in preclinical large and small tumor models

Abstract Small molecule toxin conjugates offer a novel approach to tumor antigen specific targeting, allowing reduced systemic exposures and efficient tumor penetration of cytotoxic payloads, compared to large antibody toxin conjugates. BT8009 is a Bicycle Toxin Conjugate (BTC®) in which a Nectin-4 binding Bicycle® (bicyclic peptide) is conjugated through an inert sarcosine spacer chain, and a cleavable linker, to the antimitotic toxin MMAE. Increased Nectin-4 expression has been reported in multiple tumor types (including bladder, breast, esophageal, colorectal, lung, ovarian and pancreatic cancers) and so represents an appropriate tumor binding target. Bicycles are small (1.5KDa) fully synthetic, structurally constrained peptides with the specificities and affinities akin to antibodies, and pharmacokinetic properties more like small molecules. These features make Bicycles very attractive novel drug modalities for therapeutic approaches in which low systemic exposures coupled with high local drug concentrations are desired. We have previously described the identification and optimization process leading to selection of BT8009 as a candidate for IND enabling studies. BT8009 has low nanomolar (3nM) affinity for Nectin-4 and high selectivity (>1000 fold) over Nectins 1-3 and the 5 nectin-like family members (Necl1-5). BT8009 is active in a broad range of Nectin-4 positive cell line and patient derived xenograft tumors in vivo, leading to stable diseases and tumor regressions with durable responses. This abstract describes recent work characterizing this molecule. Selectivity of the Bicycle has been further demonstrated by assessing the binding of the biotinylated version of the Nectin-4 Bicycle on a human, plasma membrane protein cell array expressing 5528 full length human plasma membrane and secreted proteins. In this assay the Bicycle bound only Nectin-4 itself, again demonstrating the excellent target specificity. Efficacy data for BT8009 has now been extended to demonstrate the anti-tumor activity of BT8009 in large (1000mm3) xenograft tumors, in which rapid and near complete responses are observed. We have further characterized the efficacy profile of BT8009 in a panel of patient-derived non-small cell lung cancer (NSCLC) xenograft models, as well as in a panel of patient-derived pancreatic ductal (PDAC) xenograft models demonstrating a high frequency of stable diseases and partial responses. We will also describe the ongoing work for identification and validation of biomarkers for treatment response. BT8009 shows excellent efficacy in xenograft models expressing Nectin-4 target and its pharmacokinetic profile enables it to provide a rapid attainment of high tumor exposure levels with reduced systemic exposure. IND-enabling studies are currently ongoing with BT8009. Citation Format: Michael Rigby, Gavin Bennett, Liuhong Chen, Gemma Mudd, Paul Beswick, Helen Harrison, Sophie Watcham, Heather Allen, Amy Brown, Katerine Van Rietschoten, Philip Jeffrey, Peter U Park, Eric Haines, Nicholas Keen, Johanna Lahdenranta. BT8009, a Bicycle Toxin Conjugate targeting Nectin-4, shows target selectivity, and efficacy in preclinical large and small tumor models [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 C061. doi:10.1158/1535-7163.TARG-19-C061

Abstract B023: A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF(V600E) colorectal tumors

Abstract Metastatic colorectal cancer (mCRC) harboring a BRAF(V600E) mutation is associated with poor prognosis and limited treatment options. Clinical trials targeting BRAF with MEK ± EGFR in 2- and 3-drug combinatorial therapy regimens have produced modest response rates and progression-free survival, with early development of resistance in most patients. Considerable efforts to study the mutational landscape or gene expression profiles or synthetic lethal genetic interactions of BRAF(V600E) mCRC have remained relatively ineffective at pinpointing novel therapeutic susceptibilities. This suggests that intrinsic resistance of BRAF(V600E) mCRC to combinatorial treatments is not driven by individual genetic dependencies, but is rather mediated by the concerted upregulation of multiple, parallel signaling pathways that cooperate to circumvent therapeutic effectiveness. To characterize which signaling circuits are rewired by BRAF and EGFR-targeted therapy, we used our new high throughput kinase activity mapping (HT-KAM) platform (Coppé et al 2019 Nature Cell Biology), which can reveal druggable kinase dependencies in cancer cell lines and tumor biospecimens by directly measuring the phospho-catalytic activity of kinases using their biological peptide targets as phospho-sensors. Out of the concerted, reprogrammed kinase circuits we identified with HT-KAM, we found a highly conserved, cell-autonomous, SRC kinases-relayed, COX2-PGE2-GNAS-driven inflammatory program that functions independently of the commonly studied BRAF-MEK-ERK / EGFR / PDPK1-AKT1 pathways. Specifically, we found that SRC family kinases are catalytically activated upon single and combination treatment with BRAF ± MEK ± EGFR inhibitors. We validated the specificity and potency of this BRAF/EGFR-independent vulnerability using genetic interventions (via shRNA or CRISPR) and drug treatments (adding dasatinib or saracatinib) in cell survival and colony formation assays across >15 BRAF(V600E) cell lines, and in patient tumor-derived xenograft (PDX) mouse models. Immunohistochemistry performed on patient tumors resistant to targeted therapies, and on residual PDX tumors after BRAF and MEK treatments, further reiterated SRC activation. Mechanistically, the activation of SRC kinases was induced by an autocrine PGE2-regulated GNAS-activation loop that COX2-inhibitors celecoxib or valdecoxib reversed both in vitro and in vivo. In PDX models, addition of celecoxib significantly improved tumor growth inhibition, and systematically outperformed 2- and 3-drug targeted therapy regimens tested in clinical trials without increasing toxicity (manuscript in preparation). In conclusion, we demonstrate that SRC-signaling is at the nexus of an autonomous inflammatory program with pro-tumorigenic activities, which may explain why BRAF(V600E) colorectal tumors develop resistance to current therapies. Moreover, targeting COX2 presents a promising new clinical strategy to restore therapeutic sensitivity in patients. Citation Format: Chloe E Atreya, Ana Ruiz-Saenz, Changjun Wang, Bo Pan, Courtney A Dreyer, Diede Brunen, Anirudh Prahallad, Danislav Spassov, Dana J Steffen, Byron C Hann, Scott R VandenBerg, Silvio Gutkind, Mark M Moasser, Laura J van 't Veer, Jean-Philippe Coppe. A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF(V600E) colorectal tumors [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 B023. doi:10.1158/1535-7163.TARG-19-B023

Preclinical PERCIST and 25% of SUVmax Threshold: Precision Imaging of Response to Therapy in Co-clinical 18F-FDG PET Imaging of Triple-Negative Breast Cancer Patient-Derived Tumor Xenografts.

Numerous recent works highlight the limited utility of established tumor cell lines in recapitulating the heterogeneity of tumors in patients. More realistic preclinical cancer models are thought to be provided by transplantable, patient-derived xenografts (PDXs). The inter- and intratumor heterogeneity of PDXs, however, presents several challenges in developing optimal quantitative pipelines to assess response to therapy. The objective of this work was to develop and optimize image metrics for 18F-FDG PET to assess response to combination docetaxel and carboplatin therapy in a co-clinical trial involving triple-negative breast cancer PDXs. We characterized the reproducibility of standardized uptake value (SUV) metrics to assess response to therapy, and we optimized a preclinical PERCIST paradigm to complement clinical standards. Considerations in this effort included variability in tumor growth rate and tumor size, solid tumors versus tumor heterogeneity and a necrotic phenotype, and optimal selection of tumor slices versus whole tumor. Methods: A test-retest protocol was implemented to optimize the reproducibility of 18F-FDG PET SUV thresholds, SUVpeak metrics, and preclinical PERCIST parameters. In assessing response to therapy, 18F-FDG PET imaging was performed at baseline and 4 d after therapy. The reproducibility, accuracy, variability, and performance of imaging metrics to assess response to therapy were determined. We defined an index called the Quantitative Response Assessment Score to integrate parameters of prediction and precision and thus aid in selecting the optimal image metric to assess response to therapy. Results: Our data suggest that a threshold of 25% of SUVmax (SUV25) was highly reproducible (<9% variability). The concordance and reproducibility of preclinical PERCIST were maximized at α = 0.7 and β = 2.8 and exhibited a high correlation with SUV25 measures of tumor uptake, which in turn correlated with the SUV of metabolic tumor. Conclusion: The Quantitative Response Assessment Score favors SUV25 followed by SUVpeak for a sphere with a volume of 14 mm3 (SUVP14) as optimal metrics of response to therapy. Additional studies are warranted to fully characterize the utility of SUV25 and preclinical PERCIST SUVP14 as image metrics for response to therapy across a wide range of therapeutic regimens and PDX models.

41P - LCSCAF1 maintains cancer stem-like traits by stabilizing c-Myc protein and promotes metastasis and recurrence in lung cancer

BackgroundAberrantly overexpressed lung cancer stem cell associated factor-X (LCSCAFX) has been implicated in non-small cell lung cancer (NSCLC) development. MethodsTo explore the effect of LCSCAFXon the lung cancer stem-like traitsin vitro and in vivo. ResultsWe reported that enforced expression of LCSCAFX promoted NSCLC cells invasion and metastasis, drug resistance and recurrencein vitro and in vivo, whereas depletion of LCSCAFX suppressed metastasis and relapse in NSCLC. Moreover, we found that LCSCAFX expression was elevated in lung cancer stem cells (LCSCs). Indeed, knockdown of LCSCAFX impaired lung cancer stem-like traits. Conversely, ectopic overexpression of LCSCAFX enhanced the self-renewal ability of lung cancer cells in vitro. In addition, elevated LCSCAFX robustly enhanced tumor initiating frequencies, as well as growth rates of NSCLC cells derived tumor xenografts in immunodeficient mice. Furthermore, mechanistic investigations established that LCSCAFX stabilized c-Myc protein by suppressing its ubiquitylation and proteasomal degradation, which is required for lung cancer stem-like properties maintenance. Consistently, immunohistochemical analysis of clinical lung tumor tissues showed that LCSCAFX was elevated in NSCLC tissues and high LCSCAFX expression serve as an independent prognostic marker in patients with NSCLC. Importantly, genetic depletion of LCSCAFX combined chemotherapy dramatically inhibited NSCLC progression in patient-derived xenograft (PDX) model. ConclusionsThese findings demonstrated a previously unappreciated role of LCSCAFX in the regulation of LCSCs, assigning LCSCAFX as a promising therapeutic target for lung cancer treatments. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

P1.12-01 CD81-HOTTIP Regulating Loop Enhances Myc-Mediated Chemoresistance in Small Cell Lung Cancer via ERK Signaling Pathway

The majority of small cell lung cancer (SCLC) patients who are treated with platinum-based chemotherapy will eventually develops into drug resistance. Currently, there are no effective durable therapies for patients with resistant recurrent SCLC. High expression of CD81 is associated with SCLC chemoresistance and poor clinical outcome. Here, our goal is to study the underlying mechanism and define a novel signaling pathway controlled by CD81 -modulating SCLC chemotherapy resistance. The expression of CD81, HOTTIP, and MYCBP were examined in human SCLC tissues, patient-derived xenograft (PDX) models, cell lines derived xenograft tumor model, and SCLC cell lines. The correlations between CD81 and HOTTIP were also confirmed by RNA immunoprecipitation and luciferase reporter assay. Functional analysis of HOTTIP, MYCBP, and ERK signaling was investigated in SCLC cell lines and xenografts. Knockdown CD81 enhanced cell responsiveness to chemotherapeutic drugs in SCLC and decreased the expression of HOTTIP, as well as inhibited the intra-nuclear translocation of MYCBP/MYC complex. Conversely, overexpression of CD81 decreased the chemotherapeutic reactivity and increased the expression of HOTTIP and promoting nuclear translocation of MYCBP/MYC complex. CD81 upregulation of HOTTIP requires the transcription factor TCF4. Further analysis demonstrated that CD81-mediated activation of ERK signaling pathway and inhibition of β-catenin degradation leads to promotion of β-catenin transfer to the nucleus. Nuclear β-catenin enhances transcriptional activity of TCF4 and then promotes the transcription of HOTTIP. Synergistically, HOTTIP could bind and increase the nucleus transduction of the MYCBP, form a HOTTIP/MYCBP/MYC complex in nucleus, resulting in increased MYC levels in nucleus. Nuclear MYC further mediates chemoresistance and promotes CD81 expression, forming a positive feedback regulatory pathway in SCLC. In addition, in vivo experiments showed that inhibitors that target the CD81-HOTTIP feedback regulatory loop combined with chemotherapeutic drugs can reverse the chemotherapeutic resistance of SCLC. Our findings suggest a novel feedback regulatory pathway dominated by CD81 and HOTTIP controlling SCLC chemotherapeutic resistance, which could serve as a new therapeutic targets for this devastating disease.

Oral administration of 3,3′-diselenodipropionic acid prevents thoracic radiation induced pneumonitis in mice by suppressing NF-kB/IL-17/G-CSF/neutrophil axis

The incidence of symptomatic radiation induced lung pneumonitis (RILP), a major dose limiting side effect of thoracic radiotherapy, is in the range of 15-40%. Therapeutic options for the prevention and treatment of RILP are limited. Hence there is a need for developing novel radioprotectors to prevent RILP which can be patient compliant. This study sought to evaluate the efficacy of oral 3,3′-diselenodipropionic acid (DSePA), a novel selenocystine derivative to prevent RILP. C3H/HeJ (pneumonitis responding) mice received a single dose of 18 Gy, whole thorax irradiation and a subset were treated with DSePA orally (2.5 mg/kg), three times per week beginning 2 h post irradiation and continued till 6 months. DSePA delayed onset of grade ≥ 2 RILP by 45 days compared to radiation control (~105 versus ~60 days). It also reversed the severity of pneumonitis in 3/10 radiation treated mice leading to significant improvement in asymptomatic survival compared to radiation control (~180 versus ~102 days). DSePA significantly (p < 0.05) reduced the radiation-mediated infiltration of polymorphonuclear neutrophils (PMN) and elevation in levels of cytokines such as IL1-β, ICAM-1, E-selectin, IL-17 and TGF-β in the bronchoalveolar lavage fluid. Moreover DSePA lowered PMN-induced oxidants, maintained glutathione peroxidase activity and suppressed NF-kB/IL-17/G-CSF/neutrophil axis in the lung of irradiated mice. Additionally, this compound did not protect A549 (lung cancer) derived xenograft tumor from radiation exposure in SCID mice. DSePA offers protection to normal lung against RILP without affecting radiation sensitivity of tumors. It has the potential to be developed as an oral agent for preventing RILP.

Abstract 3619: Nanoparticle formulation of VERU 111 for pancreatic cancer treatment

Abstract Background: Pancreatic cancer (PanCa) is one of the leading causes of cancer-related mortality in the United States due to very limited therapeutic options. Thus, developing novel therapeutic strategies will help for the management of this disease. We recently identified VERU-111, a novel synthetic molecule which showed potent anti-cancer effect against PanCa via targeting clinically important βIII and βIV tubulin isoforms. In this study, we synthesized and characterized its novel nanoformulation (MNP-VERU) and evaluated its therapeutic effects in vitro and xenograft mouse model. Methods: MNPs were prepared by chemical precipitation method and loaded with VERU-111 using diffusion method. This formulation was characterized for particle size, chemical composition, and drug loading efficiency, using various physico-chemical methods (TEM, FT-IR, DSC, TGA, and HPLC). The internalization of MNP-VERU was achieved after 6 hours incubation with MNP-VERU in PanCa cells. To determine therapeutic efficacy of MNP-VERU, we performed various in vitro (MTS, wound healing, boyden chamber real-time xCELLigence, and apoptosis assays) and in vivo (mouse tumor xenograft) studies using PanCa. Effect of MNP-VERU on various key oncogenic signaling pathways, and miRNAs was evaluated by Western blot, immunohistochemistry (IHC), confocal microscopy, qRT-PCR and in situ hybridization (ISH) analyses respectively. Result: Our novel MNP-VERU formulation provided average size of 110 nm in dynamic light scattering (DLS) and exhibited -8.23 to -11.65 mV zeta potential with an outstanding loading efficiency (94%). Cellular uptake and internalization studies demonstrate that MNP-VERU escape lysosomal degradation, providing efficient endosomal release to cytosol. MNP-VERU showed remarkable anti-cancer potential in various PanCa cells (Panc-1, AsPC-1, HPAF-II, BxPC-3, MiaPaca) and more effectively repressed βIII and βIV tubulin isoforms via restoring the expression of miR-200c. MNP-VERU more effectively suppressed AsPC-1 cells derived xenograft tumors in athymic nude mice. Conclusions: Taken together, our results suggest that MNP-VERU has more anti-cancer potential than free VERU-111 against PanCa. MNP-VERU may reduce the toxicity and improve the bioavailability of free VERU-111 and could be used for the management of PanCa. Citation Format: Vivek K. Kashyap, Bilal B. Hafeez, Qinghui Wang, Neeraj Chauhan, Prashanth K. Nagesh, Murali M. Yallapu, Duane D. Miller, Wei Li, Meena Jaggi, Subhash C. Chauhan. Nanoparticle formulation of VERU 111 for pancreatic cancer treatment [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 3619.

Combined blockade of MEK and PI3KCA as an effective antitumor strategy in HER2 gene amplified human colorectal cancer models

BackgroundTargeting the epidermal growth factor receptor (EGFR) either alone or in combination with chemotherapy is an effective treatment for patients with RAS wild-type metastatic colorectal cancer (mCRC). However, only a small percentage of mCRC patients receive clinical benefits from anti-EGFR therapies, due to the development of resistance mechanisms. In this regard, HER2 has emerged as an actionable target in the treatment of mCRC patients with resistance to anti-EGFR therapy.MethodsWe have used SW48 and LIM1215 human colon cancer cell lines, quadruple wild-type for KRAS, NRAS, BRAF and PI3KCA genes, and their HER2–amplified (LIM1215-HER2 and SW48-HER2) derived cells to perform in vitro and in vivo studies in order to identify novel therapeutic strategies in HER2 gene amplified human colorectal cancer.ResultsLIM1215-HER2 and SW48-HER2 cells showed over-expression and activation of the HER family receptors and concomitant intracellular downstream signaling including the pro-survival PI3KCA/AKT and the mitogenic RAS/RAF/MEK/MAPK pathways. HER2-amplified cells were treated with several agents including anti-EGFR antibodies (cetuximab, SYM004 and MM151); anti-HER2 (trastuzumab, pertuzumab and lapatinib) inhibitors; anti-HER3 (duligotuzumab) inhibitors; and MEK and PI3KCA inhibitors, such as refametinib and pictilisib, as single agents and in combination. Subsequently, different in vivo experiments have been performed. MEK plus PI3KCA inhibitors treatment determined the best antitumor activity. These results were validated in vivo in HER2-amplified patient derived tumor xenografts from three metastatic colorectal cancer patients.ConclusionsThese results suggest that combined therapy with MEK and PI3KCA inhibitors could represent a novel and effective treatment option for HER2-amplified colorectal cancer.

SAT-341 Pharmacology and Metabolism of GLL398 an Oral Selective Estrogen Receptor Degrader for Endocrine Therapy of Breast Cancer

Selective estrogen receptor degrader (SERD) has proven clinically effective in treating advanced or metastatic breast cancer since the approval of fulvestrant by FDA in 2002. Recent expansion of indications as a first line monotherapy and as combination therapy with CDK4/6 inhibitors further demonstrates its clinical utility as an efficacious breast cancer endocrine regimen. However, the poor pharmacokinetic properties of fulvestrant and its injection-only administration route has driven continued efforts to develop orally bioavailability SERD that could potentially improve clinical response to SERD treatment. Our laboratory has developed a boron-modified GW5638 analog, GLL398 that showed superior oral bioavailability while retaining both antiestrogenic activity and ER degrading efficacy at a potency level comparable to the more active metabolite of GW5638, GW7604. Here we report further studies on the pharmacology, pharmacokinetics, and metabolism of GLL398. Given GLL398’s robust activities in breast cancer cells that are either tamoxifen resistant or express constitutively active, mutant ESR1 (Y537S), we first measured its binding behavior towards ERY537S. To better understand the mode of action we next performed molecular modeling of the binding of GLL398 and found its molecular interactions consistent with the experimentally determined high binding affinity towards WT ER and ERY537S. To test if the superior oral bioavailability can be translated to potent efficacy in vivo, mice bearing MCF-7 derived xenograft breast tumors and patient derived xenograft tumors harboring ERY537S were treated with GLL398 which potently inhibited tumor growth in mice. Finally, the metabolism of GLL398 was also investigated to elucidate the biotransformation of the drug and its potential contribution to the superior oral bioavailability over GW7604.

Functional Role of microRNAs in Patient‐Derived Xenograft Models of Human Cholangiocarcinoma

BACKGROUND &amp; AIMSPatient‐derived xenograft (PDX) models are now being widely used in cancer research including liver and biliary carcinogenesis, and have the potential to feedback and improve the therapeutic effects in cancer patients. MicroRNAs (miRNAs) are highly conserved 19–25 nucleotide non‐coding small RNAs which are primarily involved in gene silencing. Our aim was to characterize the functional roles of microRNA regulated protein translocations in patient‐derived cholangiocarcinoma xenograft tumors.METHODSmiRNA expression in human cholangiocarcinoma cancer tissues, patient‐derived cholangiocarcinoma xenograft tissues, human extrahepatic cholangiocarcinoma cancer stem cells (GB‐CSCs)/Mz‐ChA‐1 extrahepatic cholangiocarcinoma cancer cells and their derived xenograft tumor tissues, as well as H69 non‐malignant cholangiocytes was assessed using a hybridization based microarray and was further evaluated by Taqman real‐time PCR analysis. Promoter methylation, matrix metalloproteinase (MMP) 1, 2, 3 and 9 mRNA expression was quantitated by real‐time PCR and western blot analysis.RESULTSWe identified miR‐200 family including miR‐200a, miR‐200b and miR‐200c that are differentially expressed in doxorubicin‐enriched cell fractions in a patient derived mouse xenograft tumor model of extrahepatic cholangiocarcinoma. Members of the miR‐200 family were notably more silenced in GB‐CSCs compared with their parental extrahepatic cholangiocarcinoma cancer cells (GBCs) and Mz‐ChA‐1 cells. Expression of miR‐200 family microRNAs and transmembrane protein MMP‐2 and MMP‐9 were substantially enhanced concomitant with GBCs differentiation and loss of self‐renewal, whereas forced expression of a polycistron construct encoding miR‐200 miRNAs significantly decreased chemoresistance to doxorubicin. Methylation‐specific PCR detected that miR‐200b and miR‐200c promoter was hypermethylated in GB‐CSCs, GBCs as well as Mz‐ChA‐1 cells. Cell migration and invasion were significantly decreased by miR‐200 precursor introduction in GB‐CSCs and Mz‐ChA‐1 cells. Bioinformatics and dual‐luciferase reporter assays identified ZEB1 and ZEB2 as the direct targets of miR‐200 family.CONCLUSIONOur results define a novel oncogenic regulatory mechanism of specific microRNA group in patient derived cholangiocarcinoma xenograft models and suggesting that miR‐200 cluster may be the molecular targets for eradication of human cholangiocarcinoma.Support or Funding InformationTGEN grant from Translational Genomics Research InstituteThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Poly-specific neoantigen-targeted cancer vaccines delay patient derived tumor growth

BackgroundPersonalized cancer vaccines based on neoantigens have reached the clinical trial stage in melanoma. Different vaccination protocols showed efficacy in preclinical models without a clear indication of the quality and the number of neoantigens required for an effective cancer vaccine.MethodsIn an effort to develop potent and efficacious neoantigen-based vaccines, we have developed different neoantigen minigene (NAM) vaccine vectors to determine the rules for a successful neoantigen cancer vaccine (NCV) delivered by plasmid DNA and electroporation. Immune responses were analyzed at the level of single neoantigen by flow cytometry and correlated with tumor growth. Adoptive T cell transfer, from HLA-2.1.1 mice, was used to demonstrate the efficacy of the NCV pipeline against human-derived tumors.ResultsIn agreement with previous bodies of evidence, immunogenicity was driven by predicted affinity. A strong poly-functional and poly-specific immune response was observed with high affinity neoantigens. However, only a high poly-specific vaccine vector was able to completely protect mice from subsequent tumor challenge. More importantly, this pipeline - from the selection of neoantigens to vaccine design - applied to a new model of patient derived tumor xenograft resulted in therapeutic treatment.ConclusionsThese results suggest a feasible strategy for a neoantigen cancer vaccine that is simple and applicable for clinical developments.

Molecular characterization of autophagic and apoptotic signaling induced by sorafenib in liver cancer cells.

Sorafenib is the unique accepted molecular targeted drug for the treatment of patients in advanced stage of hepatocellular carcinoma. The current study evaluated cell signaling regulation of endoplasmic reticulum (ER) stress, c-Jun-N-terminal kinase (JNK), Akt, and 5'AMP-activated protein kinase (AMPK) leading to autophagy and apoptosis induced by sorafenib. Sorafenib induced early (3-12 hr) ER stress characterized by an increase of Ser51 P-eIF2α/eIF2α, C/EBP homologous protein (CHOP), IRE1α, and sXBP1, but a decrease of activating transcription factor 6 expression, overall temporally associated with the increase of Thr183,Tyr185 P-JNK1/2/JNK1/2, Thr172 P-AMPKα, Ser413 P-Foxo3a, Thr308 P-AKt/AKt and Thr32 P-Foxo3a/Foxo3a ratios, and reduction of Ser2481 P-mammalian target of rapamycin (mTOR)/mTOR and protein translation. This pattern was related to a transient increase of tBid, Bim EL , Beclin-1, Bcl-xL, Bcl-2, autophagy markers, and reduction of myeloid cell leukemia-1 (Mcl-1) expression. The progressive increase of CHOP expression, and reduction of Thr308 P-AKt/AKt and Ser473 P-AKt/AKt ratios were associated with the reduction of autophagic flux and an additional upregulation of Bim EL expression and caspase-3 activity (24 hr). Small interfering-RNA (si-RNA) assays showed that Bim, but not Bak and Bax, was involved in the induction of caspase-3 in sorafenib-treated HepG2 cells. Sorafenib increased autophagic and apoptotic markers in tumor-derived xenograft model. In conclusion, the early sorafenib-induced ER stress and regulation of JNK and AMPK-dependent signaling were related to the induction of survival autophagic process. The sustained drug treatment induced a progressive increase of ER stress and PERK-CHOP-dependent rise of Bim EL , which was associated with the shift from autophagy to apoptosis. The kinetic of Bim EL expression profile might also be related to the tight balance between AKt- and AMPK-related signaling leading to Foxo3a-dependent BIM EL upregulation.

Synthetic Lethal Strategy Identifies a Potent and Selective TTK and CLK1/2 Inhibitor for Treatment of Triple-Negative Breast Cancer with a Compromised G1-S Checkpoint.

Historically, phenotypic-based drug discovery has yielded a high percentage of novel drugs while uncovering new tumor biology. CC-671 was discovered using a phenotypic screen for compounds that preferentially induced apoptosis in triple-negative breast cancer cell lines while sparing luminal breast cancer cell lines. Detailed in vitro kinase profiling shows CC-671 potently and selectively inhibits two kinases-TTK and CLK2. Cellular mechanism of action studies demonstrate that CC-671 potently inhibits the phosphorylation of KNL1 and SRp75, direct TTK and CLK2 substrates, respectively. Furthermore, CC-671 causes mitotic acceleration and modification of pre-mRNA splicing leading to apoptosis, consistent with cellular TTK and CLK inhibition. Correlative analysis of genomic and potency data against a large panel of breast cancer cell lines identifies breast cancer cells with a dysfunctional G1-S checkpoint as more sensitive to CC-671, suggesting synthetic lethality between G1-S checkpoint and TTK/CLK2 inhibition. Furthermore, significant in vivo CC-671 efficacy was demonstrated in two cell line-derived and one patient tumor-derived xenograft models of triple-negative breast cancer (TNBC) following weekly dosing. These findings are the first to demonstrate the unique inhibitory combination activity of a dual TTK/CLK2 inhibitor that preferably kills TNBC cells and shows synthetic lethality with a compromised G1-S checkpoint in breast cancer cell lines. On the basis of these data, CC-671 was moved forward for clinical development as a potent and selective TTK/CLK2 inhibitor in a subset of patients with TNBC. Mol Cancer Ther; 17(8); 1727-38. ©2018 AACR.