Animal Models Of Human Cancer Research Articles

Abstract 4509: Discovery of potent and highly efficacious STAT3 PROTAC degraders capable of achieving long-lasting tumor regression

Abstract STAT3 (signal transducer and activator of transcription 3) is a transcription factor and a promising therapeutic targets for cancer and other human diseases. We have previously reported the discovery of SD-36 and SD-91 as potent, selective and highly efficacious STAT3 degraders. In the present study, we report the discovery of highly potent, selective and efficacious new STAT3 degraders. These STAT3 degraders were designed using our high-affinity STAT3 ligands and high-affinity VHL-1 ligands, with UM-STAT3-3100 being the best. In direct comparison, UM-STAT3-3100 is >10-times more potent than SD-36 and SD-91 in inducing STAT3 degradation in cells and are highly selective over other STAT members in vitro and in vivo. A single intravenous administration of UM-STAT3-3100 resulted in complete and long-lasting depletion of STAT3 protein for >48 hrs in tumor and native tissues. Weekly administration of UM-STAT3-3100 at 10 mg/kg achieved complete and long-lasting tumor regression in animal models of human cancer without any signs of toxicity. UM-STAT3-3100 is a promising compound for extensive evaluation for the treatment of human cancers and other human diseases in which STAT3 plays a key role. Citation Format: Ranjan K. Acharyya, Longchuan Bai, Haibin Zhao, Dimin Wu, Metwally Hoda, McEachern Donna, Wen Bo, Duxin Sun, Shaomeng Wang. Discovery of potent and highly efficacious STAT3 PROTAC degraders capable of achieving long-lasting tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4509.

Selected Articles from This Issue

Ataxia telangiectasia mutated (ATM) kinase controls key cell cycle checkpoints and the repair of DNA double strand breaks and has been considered an attractive intervention point in DNA damage response for cancer therapy. Here, Zimmermann et al. introduce two novel ATM inhibitors. These highly potent and selective small molecules effectively suppress the ATM pathway, enhance antitumor effect of radiation and topoisomerase I inhibitors and synergize with PARP inhibitors in cancer cells and animal models of human cancer. They represent new molecular tools with potential for combination with key cancer therapies.ERα antagonists and degraders are highly effective in the treatment of the majority of ERα+ breast cancers but resistance mechanisms, including ERα mutations that facilitate ligand-independent activation, blunt the effectiveness of these therapies. Here, Furman et al. report on the development H3B-6545, a novel covalent inhibitor of ERa which demonstrates superiority over standard-of-care fulvestrant in both ERWT and ERαMUT breast tumor models. Consistent with these preclinical findings, ongoing clinical trials of H3B-6545 are demonstrating preliminary activity in ER+, HER2− metastatic breast cancer patients with both ERαWT and ERαY537S mutations.Pancreatic cancer is a devastating malignancy with a 5-year survival rate of only 9%. Glypican-1 (GPC1) is a cell surface proteoglycan that is upregulated in pancreatic cancer. Here, Ho and colleagues have constructed a GPC1-targeted immunotoxin derived from a bacterial toxin. Small molecule screening has been performed to identify irinotecan that shows synergistic efficacy with the immunotoxin. The GPC1-targeted immunotoxin inhibits pancreatic tumor growth via degradation of internalized GPC1, downregulation of Wnt signaling, and inhibition of protein synthesis. The anti-GPC1 immunotoxin in combination with irinotecan thus provides a potential new treatment for patients with pancreatic tumors.Despite the approval of immune checkpoints in small cell lung cancer (SCLC), it remains a rapidly progressing and fatal form of cancer. Recently, antibody drug conjugates (ADCs) have gained clinical traction in breast, bladder, and hematologic cancers. Here, Wiedemeyer and colleagues develop ABBV-011, a novel SEZ6-targeted, calicheamicin-based ADC that contains a non-cleavable linker which eliminates toxic metabolites associated with first generation calicheamicin ADCs. In mice, ABBV-011 potently regresses patient-derived xenografts of SCLC. ABBV-011 is being tested in a Phase 1 study and has potential as a first-in-class ADC therapy for SCLC.

TH-MYCN tumors, but not tumor-derived cell lines, are adrenergic lineage, GD2+, and responsive to anti-GD2 antibody therapy

ABSTRACT Neuroblastoma is a commonly lethal solid tumor of childhood and intensive chemoradiotherapy treatment cures ~50% of children with high-risk disease. The addition of immunotherapy using dinutuximab, a monoclonal antibody directed against the GD2 disialoganglioside expressed on neuroblasts, improves survival when incorporated into front-line therapy and shows robust activity in regressing relapsed disease when combined with chemotherapy. Still, many children succumb to neuroblastoma progression despite receiving dinutuximab-based immunotherapy, and efforts to counteract the immune suppressive signals responsible are warranted. Animal models of human cancers provide useful platforms to study immunotherapies. TH-MYCN transgenic mice are immunocompetent and develop neuroblastomas at autochthonous sites due to enforced MYCN expression in developing neural crest tissues. However, GD2-directed immunotherapy in this model has been underutilized due to the prevailing notion that TH-MYCN neuroblasts express insufficient GD2 to be targeted. We demonstrate that neuroblasts in TH-MYCN-driven tumors express GD2 at levels comparable to human neuroblastomas but rapidly lose GD2 expression when explanted ex vivo to establish tumor cell lines. This occurs in association with a transition from an adrenergic to mesenchymal differentiation state. Importantly, not only is GD2 expression retained on tumors in situ, treatment with a murine anti-GD2 antibody, 14G2a, markedly extends survival in such mice, including durable complete responses. Tumors in 14G2a-treated mice have fewer macrophage and myeloid-derived suppressor cells in their tumor microenvironment. Our findings support the utility of this model to inform immunotherapy approaches for neuroblastoma and potential opportunities to investigate drivers of adrenergic to mesenchymal fate decisions.

B7-H3-targeted Radioimmunotherapy of Human Cancer.

Targeted Radioimmunotherapy (RIT) is an attractive approach to selectively localize therapeutic radionuclides to malignant cells within primary and metastatic tumors while sparing normal tissues from the effects of radiation. Many human malignancies express B7-H3 on the tumor cell surface, while expression on the majority of normal tissues is limited, presenting B7-H3 as a candidate target for RIT. This review provides an overview of the general principles of targeted RIT and discusses publications that have used radiolabeled B7-H3-targeted antibodies for RIT of cancer in preclinical or clinical studies. Databases including PubMed, Scopus, and Google Scholar were searched for publications through June 2018 using a combination of terms including "B7-H3", "radioimmunotherapy", "targeted", "radiotherapy", and "cancer". After screening search results for relevancy, ten publications were included for discussion. B7-H3-targeted RIT studies to date range from antibody development and assessment of novel Radioimmunoconjugates (RICs) in animal models of human cancer to phase II/III trials in humans. The majority of clinical studies have used B7-H3-targeted RICs for intra- compartment RIT of central nervous system malignancies. The results of these studies have indicated high tolerability and favorable efficacy outcomes, supporting further assessment of B7-H3-targeted RIT in larger trials. Preclinical B7-H3-targeted RIT studies have also shown encouraging therapeutic outcomes in a variety of solid malignancies. B7-H3-targeted RIT studies over the last 15 years have demonstrated feasibility for clinical development and support future assessment in a broader array of human malignancies. Future directions worthy of exploration include strategies that combine B7-H3- targeted RIT with chemotherapy or immunotherapy.

Discovery of Highly Potent, Selective, and Orally Efficacious p300/CBP Histone Acetyltransferases Inhibitors

p300 and CREB-binding protein (CBP) are ubiquitously expressed pleiotropic lysine acetyltransferases and play a key role as transcriptional co-activators that are essential for a multitude of cellular processes. Despite great importance, there is a lack of highly selective, potent, druglike p300/CBP inhibitors. Through the artificial-intelligence-assisted drug discovery pipeline and further optimization, we reported the discovery of novel, highly selective, potent small-molecule inhibitors of p300/CBP histone acetyltransferases (HAT) with desired druglike properties, exemplified by B026. Our data demonstrated that B026, with half maximal inhibitory concentration (IC50) values of 1.8 nM to p300 and 9.5 nM to CBP enzyme inhibitory activity, is the most potent, selective p300/CBP HAT inhibitor. Moreover, B026 achieves significant and dose-dependent tumor growth inhibition in an animal model of human cancer, suggesting that B026 is a highly promising p300/CBP HAT inhibitor and warrants extensive preclinical investigation as a potential clinical development candidate.

Preclinical Applications of Magnetic Resonance Imaging in Oncology.

The evolving possibilities of molecular imaging (MI) are fundamentally changing the way we look at cancer, with imaging paradigms now shifting away from basic morphological measures toward the longitudinal assessment of functional, metabolic, cellular, and molecular information in vivo. Recent developments of imaging methodology and probe molecules utilizing the vast number of novel animal models of human cancers have enhanced our ability to non-invasively characterize neoplastic tissue and follow anticancer treatments. While preclinical molecular imaging offers a whole palette of excellent methodology to choose from, we will focus on magnetic resonance imaging (MRI) techniques, since they provide excellent molecular imaging capabilities and bear high potential for clinical translation. Prerequisites and consequences of using animal models as surrogates of human cancers in preclinical molecular imaging are outlined. We present physical principles, values, and limitations of MRI as molecular imaging modality and comment on its high potential to non-invasively assess information on metabolism, hypoxia, angiogenesis, and cell trafficking in preclinical cancer research.

Methods for Evaluation of a Snake Venom-Derived Disintegrin in Animal Models of Human Cancer.

Integrin targeting has been shown to be an effective approach for anticancer therapy. We engineered a recombinant disintegrin, vicrostatin (VCN), that binds with high affinity and specificity to the Arg-Gly-Asp (RGD) class of integrins, including αvβ3, αvβ5, and α5β1, involved in tumor invasion and metastasis. We used three different delivery modalities to examine anticancer activity of VCN in mouse models of human ovarian cancer, glioma, and prostate cancer. A female mouse model was used to examine the treatment of established ovarian cancer (OC) using VCN delivered intraperitoneally (IP) weekly either in saline or impregnated in a viscoelastic gel. SKOV3luc cells (a human OC cell line) were directly injected IP into immunodeficient mice. We also examined the antitumor activity of radioiodinated VCN delivered intravenously in a human glioma model in nude mice. We evaluated the effectiveness of 131I-VCN in combination with the DNA alkylating agent temozolomide in limiting glioma growth. Finally, treatment of a bone metastatic model of human prostate cancer (PC) in immunodeficient mice was examined using a liposomal formulation of VCN (LVCN) delivered intravenously. Human PC cells were suspended in a solution of Matrigel and injected into the left tibia of immunodeficient mice. Diameters of both the left and right (control) tibias were measured by caliper repeatedly after VCN treatment was initiated.

Next-Generation in vivo Modeling of Human Cancers.

Animal models of human cancers played a major role in our current understanding of tumor biology. In pre-clinical oncology, animal models empowered drug target and biomarker discovery and validation. In turn, this resulted in improved care for cancer patients. In the quest for understanding and treating a diverse spectrum of cancer types, technological breakthroughs in genetic engineering and single cell “omics” offer tremendous potential to enhance the informative value of pre-clinical models. Here, I review the state-of-the-art in modeling human cancers with focus on animal models for human malignant gliomas. The review highlights the use of glioma models in dissecting mechanisms of tumor initiation, in the retrospective identification of tumor cell-of-origin, in understanding tumor heterogeneity and in testing the potential of immuno-oncology. I build on the deep review of glioma models as a basis for a more general discussion of the potential ways in which transformative technologies may shape the next-generation of pre-clinical models. I argue that refining animal models along the proposed lines will benefit the success rate of translation for pre-clinical research in oncology.

Abstract 5109: Somatic CRISPR/Cas9-mediated gene editing enables versatile brain tumor modeling

Abstract Despite substantial advances in the molecular classification of pediatric brain tumors over the last decade, many tumors are still incurable, have a poor prognosis or therapy-related detrimental long term effects. To develop innovative and modern therapy approaches, sophisticated mouse models, faithfully reflecting the human disease are urgently needed. However, the development of transgenic mouse lines is a time consuming process that is confronted with an increasing number of candidate genes resulting from next generation sequencing of tumor specimens. To cope with this discrepancy, more adaptable animal models are needed. Using somatic gene transfer to manipulate cells in situ holds great advantages in terms of speed and flexibility. We therefore established a system in which we are using the CRISPR/Cas9 system for somatic disruption of candidate tumor suppressor genes (TSGs) in combination with transposon-mediated overexpression of candidate oncogenes to induce murine brain tumors. By deleting Ptch1 in murine cerebella, we induced Sonic Hedgehog (SHH) subgroup medulloblastoma with high penetrance and short latency. We characterized the induced mutations in depth and did not detect recurrent off-targets using unsupervised whole genome sequencing. We further demonstrated that this method is suited to induce other brain tumor entities by targeting multiple TSGs at the same time or by combining TSG targeting with overexpression of certain oncogenes. To date we established five novel mouse models using this system. Two of these resulted from simultaneous deletion of Nf1, Pten and Trp53 or from combining overexpression of a MET fusion with a loss of Trp53, respectively. Both of these combinations led to efficient glioblastoma induction in the murine forebrain. Our results demonstrate a fast and flexible system for validating novel candidate genes and for generating faithful animal models of human cancer. Citation Format: Marc Zuckermann, Britta Ismer, Volker Hovestadt, Christiane B. Knobbe-Thomsen, Marc Zapatka, Paul A. Northcott, Martine F. Roussel, Guido Reifenberger, Peter Lichter, Stefan M. Pfister, Daisuke Kawauchi, Jan Gronych, David T. Jones. Somatic CRISPR/Cas9-mediated gene editing enables versatile brain tumor modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5109.

Abstract A01: Somatic engineering of the mammary gland for the development of novel mouse models of triple-negative breast cancer

Abstract Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. For these reasons, new technologies are needed to expand the genetic toolbox of cancer biologists and allow a more rapid and systematic in vivo interrogation of gene perturbations. In this regard, the advent of CRISPR/Cas9 technologies for somatic genome editing has already paved the way for a new generation of non-germline animal tumor models. For example, liver-specific gene disruption was achieved by transient delivery of components of the CRISPR/Cas9 system in the tail veins of mice, leading to hepatocellular carcinoma (Xue et al., 2014; Weber et al., 2015). Similar approaches have been used to deliver targeted oncogenic mutations to the lung (Platt et al., 2014; Sánchez-Rivera et al., 2014), brain (Zuckermann et al., 2015), and pancreas (Chiou et al., 2015). We have previously shown that the mammary tissue is also amenable to somatic gene editing using intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in the mammary glands of female mice carrying conditional predisposing alleles (Annunziato et al., 2016). This approach was successfully applied to validate in vivo candidate tumor suppressors implicated in invasive lobular carcinoma (Kas et al., 2017), but it is conceivable that more breast cancer subtypes can be modeled via somatic engineering in mice with distinct predisposing mutations. In this study, we show how somatic engineering via intraductal injection of lentiviruses can be used to develop innovative mouse models of triple-negative breast cancer (TNBC), one of the subtypes with the poorest prognosis in the clinics. Using intraductal injection of Cre-encoding lentiviruses in mice with conditional Brca1 and Trp53 alleles (B1P), we were able to target TNBC-initiating cells from the basal compartment, and induce tumors that retained all the hallmark features of the transgenic Cre-driven tumor counterparts, including latency, histopathology, and DNA copy-number variation (CNV) pattern. Moreover, we could dramatically accelerate tumorigenesis when lentiviral Cre was injected in B1PM mice that harbored, in addition to conditional Brca1 and Trp53 alleles, a conditional knock-in allele overexpressing the Myc oncogene, a candidate driver in BRCA1-associated cancers. Alternatively, somatic Myc expression and loss of BRCA1 and p53 was induced in the mammary glands of B1P mice via intraductal injection of lentiviral vectors encoding Myc and Cre under a viral promoter. In both cases the resulting tumors displayed a reshaped CNV profile that strongly implicated a limited number of recurrent focal amplifications and deletions in the tumorigenic cascade. We reiterated the process by lentiviral overexpression of these candidate driver oncogenes and Cre in the mammary glands of B1PM mice, or by achieving in situ CRISPR/Cas9-mediated somatic gene disruption of one or multiple candidate tumor-suppressor genes in Cas9-transgenic WB1P-Cas9 mice. This versatile somatic platform allows for rapid, flexible, and multiplexable in vivo testing of putative oncogenic hits and deals with the need for increasingly complex mouse models while avoiding the bottlenecks of classical transgenesis. Notably, we show here that we can extend the applicability of non-germline breast cancer modeling in mice to produce genetically and histologically accurate TNBC, which allows in vivo investigation of the underlying biology as well as rapid generation of tailored preclinical models for this aggressive breast cancer subtype. Citation Format: Stefano Annunziato, Julian R. de Ruiter, Chiara S. Brambillasca, Sjors M. Kas, Federica Ferrante, Catrin Lutz, Bjorn Siteur, Bas van Gerwen, Marieke van de Ven, Martine H. van Miltenburg, Linda Henneman, Jos Jonkers. Somatic engineering of the mammary gland for the development of novel mouse models of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A01.

Therapeutic TCR Mimic Monoclonal Antibodies to Intracellular Oncogenic Proteins

Nearly all tumor-specific targets and oncogenic proteins are intracellular proteins. Few are druggable with current small molecules. Neither mAb nor mAb-directed CAR T cells can enter the cell to recognize these proteins. In contrast, naturally occurring T cells are evolved to recognize MHC-presented peptide epitopes from intracellular proteins. These are generally derived from intracellular proteins. Therefore, there is a need to create antibodies that recognize antigens from intracellular, tumor specific and oncogenic proteins. Phage display libraries were used to create TCR mimic (TCRm) monoclonal antibodies (mAb) to peptide/MHC complexes presenting peptides from important intracellular proteins. The targets are peptide/MHC complexes from: the onco-fetal antigen WT1 protein and the cancer-testis antigen “preferentially expressed antigen in melanoma” (PRAME), both of which are overexpressed in multiple neoplasms, making them a highly attractive therapeutic targets, FoxP3, a marker and regulator of Treg cells, and other oncogenic proteins. The TCRm were studied in vitro and in animal models. TCRm to low-density peptide epitopes from undruggable intracellular proteins presented in the context of MHC molecules were made and are therapeutically effective in mouse models of human cancers. Human IgG1, Fc region afucosylated forms with enhanced FcR binding, bispecific mAb and BiTEs, as well as CAR T cells, were made with these TCRm. The efficacy of these TCRm may be limited by the ultra-low density of the target epitopes, which can be 50-100 times below that of typical antigenic targets of therapeutic antibodies. CD47 blockade by use of a high affinity SIPRα variant protein (CV1) has been shown to improve the effects of mAb by enhancement of antibody dependent cellular phagocytosis. CV1 with TCRm together were superior in combination therapy as compared to either drug alone in suppressing leukemia growth in the liver, spleen and bone marrow and prompting long-term survival benefits. New mechanisms of action, issues related to specificity, and reasons for escape from the actions of TCRm will be described. TCRm represent a new class of mAb with unusual specificity and mechanisms of actions. Several are therapeutically active in different formats in animal models of human cancer.

Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland.

Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice.

Anti-Cancer Effect of Cap-Translation Inhibitor 4EGI-1 in Human Glioma U87 Cells: Involvement of Mitochondrial Dysfunction and ER Stress

Background: Cancer cells are frequently addicted to deregulated oncogenic protein translation that usually arises as a consequence of increased signaling flux from eIF4F activation. The small molecule 4EG-I, a potent inhibitor of translation initiation through disrupting eIF4E/eIF4G interaction, has been shown to exert anticancer effects in animal models of human cancers. Methods: Here, we extensively investigated the anticancer activity of 4EGI-1 in human glioma U87 cells. The anti-cancer effects of 4EGI-1 were measured by cell viability, lactate dehydrogenase (LDH) release, TUNEL staining, flow cytometry and western blot analysis in vitro, and also examined in a U87 xenograft model in vivo. The potential underlying molecular mechanisms were investigated by measuring mitochondrial function and ER stress. Results: We found that 4EGI-1 impaired the assembly of the eIF4F complex and decreased the expression of the eIF4E regulated proteins. The results of TUNEL staining and flow cytometry showed that 4EGI-1 treatment induced apoptotic cell death in a dose-dependent manner. Furthermore, 4EGI-1-induced apoptosis in U87 cells was associated with mitochondrial dysfunction and activation of the intrinsic mitochondrial pathway, which was dependent on the induction of the pro-apoptotic protein Bax. In addition, 4EGI-1 treatment triggered ER stress, which was evidenced by morphological changes of ER lumen and ER calcium release, as well as the dose-dependent increases in the expression of ER stress related proteins. Moreover, knockdown of the ER chaperone GRP-78 through siRNA was shown to partially reverse the 4EGI-1-induced ER stress in U87 cells. In vivo, 4EGI-1 strongly inhibited growth of U87 glioma xenografts without any apparent organ related toxicities. Conclusion: These data indicate that the use of inhibitors that directly target the translation initiation complex eIF4F could represent a potential novel approach for human glioma therapy.

A GFP-Tagged Gross Deletion on Chromosome 1 Causes Malignant Peripheral Nerve Sheath Tumors and Carcinomas in Zebrafish.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft-tissue sarcomas, characterized by complex karyotypes. The molecular bases of such malignancy are poorly understood and efficient targeted molecular therapies are currently lacking. Here we describe a novel zebrafish model of MPNSTs, represented by the transgenic mutant line Tg(-8.5nkx2.2a:GFP) ia2. ia2 homozygous animals displayed embryonic lethality by 72 hpf, while the heterozygotes develop visible tumor masses with high frequency in adulthood. Histological and immunohistochemical examination revealed aggressive tumors with either mesenchymal or epithelial features. The former (54% of the cases) arose either in the abdominal cavity, or as intrathecal/intraspinal lesions and is composed of cytokeratin-negative spindle cells with fascicular/storiform growth pattern consistent with zebrafish MPNSTs. The second histotype was composed by polygonal or elongated cells, immunohistochemically positive for the pan-cytokeratin AE1/AE3. The overall histologic and immunohistochemical features were consistent with a malignant epithelial neoplasm of possible gastrointestinal/pancreatic origin. With an integrated approach, based on microsatellite (VNTR) and STS markers, we showed that ia2 insertion, in Tg(-8.5nkx2.2a:GFP) ia2 embryos, is associated with a deletion of 15.2 Mb in the telomeric portion of chromosome 1. Interestingly, among ia2 deleted genes we identified the presence of the 40S ribosomal protein S6 gene that may be one of the possible drivers for the MPNSTs in ia2 mutants. Thanks to the peculiar features of zebrafish as animal model of human cancer (cellular and genomic similarity, transparency and prolificacy) and the GFP tag, the Tg(-8.5nkx2.2a:GFP) ia2 line provides a manageable tool to study in vivo with high frequency MPNST biology and genetics, and to identify, in concert with the existing zebrafish MPNST models, conserved relevant mechanisms in zebrafish and human cancer development.

Abstract PR02: Somatic CRISPR/Cas9-mediated tumor suppressor disruption enables versatile brain tumor modeling

Abstract Modeling cancer in mice through engineering of candidate genes in the germline has long been the gold standard for the validation of putative oncogenes or tumor suppressor genes (TSGs). For TSGs, whereby loss-of-function (LOF) mutations act as a driver for malignant transformation, this has traditionally been accomplished using constitutive or cell type-specific knockout strategies mediated by homologous recombination in embryonic stem cells. While this allows evaluation of cell type-specific susceptibility to malignant transformation, generation of genetically engineered mouse models (GEMMs) is a time consuming process. For in vivo investigation of a large number of molecular alterations, such as the many new candidates currently emerging from large-scale tumor genome sequencing efforts, a faster and more flexible method is required. We therefore sought to adapt the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-guided endonuclease technique for the somatic disruption of candidate TSGs and thereby complement aforementioned already existing models like GEMM. To provide a flexible and effective method for investigating somatic LOF alterations and their influence on tumorigenesis in vivo, we established in vivo somatic gene transfer of CRISPR/Cas9-encoding vectors using polyethylenimine or in utero electroporation, respectively, allowing for in vivo targeting of TSGs in the developing murine brain. We demonstrate the utility of this approach by deleting the tumor suppressor Ptch1, which resulted in the development of cerebellar tumors resembling sonic hedgehog (SHH) subgroup medulloblastoma both at the histopathological as well as the molecular level. Furthermore, we show that multiple genes can be disrupted with this approach, using in utero electroporation of guide RNAs (gRNAs) targeting Trp53, Pten and Nf1 into the forebrain of mice. This resulted in induction of glioblastoma with 100% penetrance. Using whole genome sequencing (WGS) we characterized the MB-driving Ptch1 deletions in detail and show that no off-targets were detected in these tumors. Taken together, these approaches provide a fast and convenient way for validating the emerging wealth of novel candidate TSGs and the generation of faithful animal models of human cancer. Citation Format: Marc Zuckermann, Volker Hovestadt, Christiane B. Knobbe-Thomsen, Marc Zapatka, Paul A. Northcott, Kathrin Schramm, Jelena Belic, David T.W. Jones, Barbara Tschida, Branden Moriarity, David Largaespada, Martine F. Roussel, Andrey Korshunov, Guido Reifenberger, Stefan M. Pfister, Peter Lichter, Daisuke Kawauchi, Jan Gronych. Somatic CRISPR/Cas9-mediated tumor suppressor disruption enables versatile brain tumor modeling. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr PR02.

Growth hormone, insulin-like growth factor system and carcinogenesis.

The growth hormone (GH) and insulin-like growth factor (IGF) system plays an important role in the regulation of cell proliferation, differentiation, apoptosis, and angiogenesis. In terms of cell cycle regulation, the GH-IGF system induces signalling pathways for cell growth that compete with other signalling systems that result in cell death; thus the final effect of these opposed forces is critical for normal and abnormal cell growth. The association of the GH-IGF system with carcinogenesis has long been hypothesised, mainly based on in vitro studies and the use of a variety of animal models of human cancer, and also on epidemiological and clinical evidence in humans. While ample experimental evidence supports a role of the GH-IGF system in tumour promotion and progression, with several of its components being currently tested as central targets for cancer therapy, the strength of evidence from patients with acromegaly, GH deficiency, or treated with GH is much weaker. In this review, we will attempt to consolidate this data. (Endokrynol Pol 2016; 67 (4): 414-426).

Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling.

In vivo functional investigation of oncogenes using somatic gene transfer has been successfully exploited to validate their role in tumorigenesis. For tumour suppressor genes this has proven more challenging due to technical aspects. To provide a flexible and effective method for investigating somatic loss-of-function alterations and their influence on tumorigenesis, we have established CRISPR/Cas9-mediated somatic gene disruption, allowing for in vivo targeting of TSGs. Here we demonstrate the utility of this approach by deleting single (Ptch1) or multiple genes (Trp53, Pten, Nf1) in the mouse brain, resulting in the development of medulloblastoma and glioblastoma, respectively. Using whole-genome sequencing (WGS) we characterized the medulloblastoma-driving Ptch1 deletions in detail and show that no off-targets were detected in these tumours. This method provides a fast and convenient system for validating the emerging wealth of novel candidate tumour suppressor genes and the generation of faithful animal models of human cancer.

Targeted therapy against cancer stem cells.

Research into cancer stem cells (CSCs), which have the ability to self-renew and give rise to more mature (differentiated) cancer cells, and which may be the cells responsible for the overall organization of a tumor, has progressed rapidly and concomitantly with recent advances in studies of normal tissue stem cells. CSCs have been reported in a wide spectrum of human tumors. Like normal tissue stem cells, CSCs similarly exhibit significant phenotypic and functional heterogeneity. The ability of CSCs to self-renew results in the immortality of malignant cells at the population level, whereas the ability of CSCs to differentiate, either fully or partially, generates the cellular hierarchy and heterogeneity commonly observed in solid tumors. CSCs also appear to have maximized their pro-survival mechanisms leading to their relative resistance to anti-cancer therapies and subsequent relapse. Studies in animal models of human cancers have also provided insight into the heterogeneity and characteristics of CSCs, helping to establish a platform for the development of novel targeted therapies against specific CSCs. In the present study, we briefly review the most recent progress in dissecting CSC heterogeneity and targeting CSCs in various human tumor systems. We also highlight a few examples of CSC-targeted drug development and clinical trials, with the ultimate aim of developing more effective therapeutic regimens that are capable of preventing tumor recurrence and metastasis.

Quantifying the lower limit of antibody-dependent cell cytotoxicity using a TCRm against a WT1 peptide/MHC epitope (VAC3P.1058)

Abstract The WT1 protein is an ideal cancer antigen, but therapeutically targeting this transcription factor has proven difficult. We have discovered a therapeutic TCR mimic antibody that specifically binds to a WT1-derived peptide (RMFPNAPYL) in the context of the MHC class I haplotype HLA-A2. This high affinity mAb, called ESK, has demonstrated significant anti-cancer activity in animal models of human cancers and leukemias. Studies in vitro and in vivo have shown the dominant role of ADCC in ESK activity. While commercially available mAbs target tens to hundreds of thousands of sites on a cell, ESK binds as few as ~500 epitopes and still promotes cancer cell clearance in vivo. Therefore, we believe ADCC can be a more sensitive mode of killing than currently estimated. To probe this question, we developed a biotinylated RMF peptide that can be pulsed onto HLA-A2 on T2 cells at various concentrations and detected using labeled streptavidin probes. By using 3D confocal microscopy photon scanning, we can detect single molecules of the streptavidin probe on a cell surface. Therefore, using a CFP-DEVD-YFP FRET reporter expressed in the pulsed T2 cells, we can measure the number of peptides bound by ESK in the immunological synapse between a target T2 cell and a human NK cell required to induce T2 cell death. Identifying this number will impact patient selection for ESK clinical trials and will influence the development of therapeutic mAbs against other low-density epitopes.

Dual targeting of the tumor and its associated vasculature using a single bispecific chimeric antigen receptor molecule

Meeting abstracts We have previously demonstrated the efficacy of HER2 specific chimeric antigen receptor (CAR) T cells in animal models of human cancer. While HER2 CAR T cells induced tumor regression, tumors recurred in a subset of animals. Lytic co-targeting of the tumor endothelium could