Genetically-modified Mouse Models Research Articles

Abstract 7553: Dual inhibition of chromatin remodeling and DNA methylation as a novel treatment for triple negative breast cancer

Abstract Epigenetics plays a central role in the initiation, establishment, and progression of cancer. In this capacity, epigenetics regulates several abnormal nuclear processes in the cancer cell including DNA replication, DNA damage repair and most prominently transcription. Because of their reversibility, epigenetic modifications have been aggressively pursued pharmacologically to correct abnormal cancer biology in the hopes of reversing cancer phenotypes. To develop novel epigenetic based therapeutic strategies for triple negative breast cancer (TNBC) we combined the DNA methyltransferase inhibitor (DNMTi) guadecitabine (Gua) with a genetic CRISPR/Cas9 mediated knockout (KO) of the chromatin remolding protein Bromodomain PHD-finger Transcription Factor (BPTF). Using the E0771 and 4T1 mouse TNBC models we show that BPTF depleted tumor cells are more susceptible to the antitumor effects of Gua. These antitumor effects are not observed in culture, or in immune compromised NSG mice, suggesting that their effects on cancer cell growth are not cancer cell intrinsic. RNA-Seq, ATAC-Seq and MBD-Seq analysis from FACS purified cancer cells shows that the combination of BPTF inhibition and Gua alters immune regulatory cytokine expression (BMP4 and CSF3) in the cancer cell, and it is theorized that these changes alter the tumor microenvironment to become less immune suppressive and permissive to the antitumor effects of immune cells. This hypothesis is supported by Cytek phenotyping of tumor resident and peripheral immune cells from treated mice showing reduced levels of immune suppressive myeloid derived suppressor cells (MDSC). Inhibiting BPTF pharmacologically using the bromodomain inhibitor AU1 does not recapitulate the effects of the genetic inhibition suggesting that the benefits of BPTF depletion to sensitization to Gua are not captured by inhibition of its bromodomain. To model the effects of pharmacologic BPTF inhibition we created a BPTF mouse KO genetically modified mouse model (GEMM). We show using this GEMM that BPTF KO in the adult mouse (a tamoxifen inducible Cre) is not lethal and has beneficial effects to the control of E0771 tumors, possibly through depleting tumor resident naïve T cells, and that transplanting BPTF KO E0771 tumors into BPTF KO adult mice further improves tumor growth control. These benefits in tumor growth control are prevented with CD8 depletion. These results in total suggests that a combination therapy regimen including BPTF inhibition (a PROTAC degradation approach) with a DNMTi could provide clinical benefits to patients with TNBC through both tumor cell intrinsic and extrinsic mechanisms. Citation Format: Joseph W. Landry, Jubin Kang, Christiane Morecock, Harry Bear, Rebecca Martin. Dual inhibition of chromatin remodeling and DNA methylation as a novel treatment for triple negative breast cancer [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 7553.

Abstract A061: A novel genetically engineered mouse model of Myc-driven pancreatic cancer recapitulates phenotypic heterogeneity, metastasis, and therapy resistance seen in clinical populations

Abstract One of the most aggressive cancers in the US is pancreatic ductal adenocarcinoma (PDAc), whose incidence is expected to rise in the coming years. The standard-of-care treatments have improved over the past few decades, but they still primarily involve harsh chemotherapy cocktails and have only marginally increased overall survival, to a 5-year survival rate of 12 percent. c-MYC (MYC), a potent transcription factor, is frequently dysregulated in PDAc. In PDAc and other cancers, MYC plays a significant role in tumor progression, and its dysregulation has been linked to tumor aggressiveness and therapeutic resistance. In various mouse models of cancer, it has been demonstrated that oncogenic MYC expression controls components of the tumor microenvironment (TME). MYC expression in PDAc has been associated with a desmoplastic immune suppressive TME, though the precise mechanism is still unknown. In this study, we introduce a novel genetically modified mouse model (GEMM) of PDAc that can be used to simulate the disease's heterogeneity and investigate how MYC controls the tumor’s immune and stromal microenvironments. In the pancreas of our KMCERT2 model, both mutant Kras and lowly deregulated Myc are expressed through inducible Cre-driven mechanisms. Our research demonstrates how deregulated MYC cooperates with oncogenic KRASG12D in the adult pancreas to promote PDAc, faithfully reproducing the inter- and intra-tumoral heterogeneity observed in clinical PDAc populations. Currently, an embryonic KrasG12D- and p53 loss/mutant-driven PDAc model (KPC) is used for the majority of murine studies of PDAc. To learn more about the mechanisms underlying observed phenotypes in our KMCERT2 model, RNA- and DNA sequencing were performed on microdissected autochthonous tumor specimens as well as KMCERT2 tumor-derived cell lines. In contrast to the KPC model, our inducible KMCERT2 model of PDAc exhibits genetic alterations, such as CDKN2A loss, comparable to human disease. A distinct loss of nuclear p53 expression is also observed. Intriguingly, multiplexed immunohistochemistry analysis of immune cell composition showed that MYC-driven tumors have a higher density of antigen-presenting cells (APCs) when compared to spontaneous KPC tumors. Standard-of-care medications like gemcitabine and FOLFIRINOX are frequently ineffective against human PDAc. Orthotopic therapeutic studies using our KMCERT2 tumor-derived cell lines show a variety of responses to these therapies, with both resistant and sensitive tumor models, allowing us to use this model to better understand the mechanisms causing therapeutic resistance and to test new therapies. Additionally, we observe consistent liver metastasis in both spontaneous and orthotopic transplant settings. Together, these studies investigate the function of dysregulated MYC expression in murine PDAc metastatic behavior, immune phenotypes, and therapeutic response. It also offers mouse models of PDAc that are both spontaneous and orthotopic, recapitulating the complex and highly metastatic nature of the human disease. Citation Format: Isabel A. English, Patrick J. Worth, Kevin A. MacPherson, Michael B. Heskett, Vidhi Shah, Jackie L. Phipps, Courtney Betts, Shamilene Sivagnanam, Carl Pelz, Amy S. Farrell, Brittany L. Allen-Petersen, Meghan M. Joly, Motoyuki Tsuda, Colin J. Daniel, Jenny Eng, Xiaoyan Wang, Ashley L. Kiemen, Laura D. Wood, Terry K. Morgan, Mary C. Thoma, Jennifer M. Chu, Paul T. Spellman, Lisa M. Coussens, Ellen M. Langer, Rosalie C. Sears. A novel genetically engineered mouse model of Myc-driven pancreatic cancer recapitulates phenotypic heterogeneity, metastasis, and therapy resistance seen in clinical populations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A061.

Abstract B089: Oncogenic signaling and responses to treatment in RAS mutant cancers are mutation-specific

Abstract The KRAS, NRAS, and HRAS genes are frequently mutated in cancer, often at hotspots altering codons 12, 13, and 61. Yet how different RAS isoforms and mutations precisely regulate oncogenic signaling is still largely unknown. For example, the role of mutant RAS in driving overactivation of ERK signaling in cancer has long been elucidated, but its involvement in the oncogenic activation of PI3K is still controversial, and different studies have reported conflicting findings regarding the requirement of RAS for physiological and oncogenic PI3K signaling. One of the reasons for this lack of understanding has been the inability to directly target RAS, which has historically been circumvented through genomic perturbation and murine models. However, these approaches are limited because it is not possible to distinguish between the direct effects of RAS on PI3K and compensatory events that occur as an adaptation to the oncogene. With the discovery of RAS inhibitors, we can now directly interrogate the biological effects of RAS isoforms and hotspot mutations in cancer cells with improved time resolution. Here, we use RM-042, a first-in-class broad-spectrum RAS·GTP inhibitor (RASMULTI(ON)), to characterize the features and therapeutic vulnerabilities of mutant RAS signaling in human cancers. Through a detailed biochemical analysis of the signaling response to RAS and MEK inhibition, we reveal that RAS-mediated oncogenic signaling is largely independent of the mutant RAS isoform, rather depending on the mutant codon. Specifically, RASQ61X is exquisitely sensitive to MAPK pathway inhibition, confers near-complete independence of upstream regulation, and is impaired in its signaling to PI3K/Akt/mTOR. Conversely, RASG12X enables oncogenic signaling to both MAPK and PI3K/Akt/mTOR by amplifying upstream input from receptor tyrosine kinases (RTKs). Simultaneous RAS and RTK inhibition selectively impairs the growth of RASG12X-mutant tumors, irrespective of RAS isoform and tumor type, and we investigate the molecular mechanisms behind this synergistic effect. Additionally, we have generated a genetically modified mouse model (GEMM) harboring liver tumors induced by RASG12D or RASQ61R. In cell lines derived from these respective models, RASG12D but not RASQ61R activates PI3K upon growth-factor stimulation. Furthermore, in this system, RASQ61R is less oncogenic than RASG12D, an effect that can be rescued by the concurrent deletion of PTEN. These findings provide mechanistic insights into how mutant RAS interacts with upstream and downstream partners to maximize oncogenic signaling. Furthermore, our results shed light on the selective pressure driving the emergence of RASQ61X mutations as a mechanism of resistance against RTK inhibition: we show that colorectal cancer cells that acquire RASQ61X mutations to overcome EGFR inhibition become more independent of upstream signaling than those that acquire RASG12X. Citation Format: Michelangelo Marasco, Dinesh Kumar, Riccardo Mezzadra, Wei Luan, Ilinca Aricescu, Rona Yaeger, Vladimir Markov, Yu Zhu, Elisa de Stanchina, Scott Lowe, Sandra Misale, Neal Rosen. Oncogenic signaling and responses to treatment in RAS mutant cancers are mutation-specific [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B089.

High-density lipoproteins and non-alcoholic fatty liver disease

Background and aimsNon-alcoholic fatty liver disease (NAFLD), a high incidence liver pathology, is associated with a ∼1.5-fold higher cardiovascular disease risk. This phenomenon is generally attributed to the NAFLD-associated increase in circulating levels of pro-atherogenic apolipoprotein B100-containing small dense low-density lipoprotein and plasma hypertriglyceridemia. However, also a significant reduction in cholesterol transported by anti-atherogenic high-density lipoproteins (HDL) is frequently observed in subjects suffering from NAFLD as compared to unaffected people. In this review, we summarize data regarding the relationship between NAFLD and plasma HDL-cholesterol levels, with a special focus on highlighting potential causality between the NAFLD pathology and changes in HDL metabolism. Methods and resultsPublications in PUBMED describing the relationship between HDL levels and NAFLD susceptibility and/or disease severity, either in human clinical settings or genetically-modified mouse models, were critically reviewed for subsequent inclusion in this manuscript. Furthermore, relevant literature describing effects on lipid loading in cultured hepatocytes of models with genetic alterations related to HDL metabolism have been summarized. ConclusionsAlthough in vitro observations suggest causality between HDL formation by hepatocytes and protection against NAFLD-like lipid accumulation, current literature remains inconclusive on whether relative HDL deficiency is actually driving the development of fatty liver disease in humans. In light of the current obesity pandemic and the associated marked rise in NAFLD incidence, it is of clear scientific and societal interest to gain further insight into the relationship between HDL-cholesterol levels and fatty liver development to potentially uncover the therapeutic potential of pharmacological HDL level and/or function modulation.

EPEN-06. CHARACTERISING THE DEVELOPMENTAL ORIGINS AND TRAJECTORIES OF ZFTA-RELA FUSION EPENDYMOMA IN A NOVEL MOUSE MODEL

Abstract Ependymomas driven by the ZFTA-RELA fusion account for >70% of all supratentorial ependymomas. These tumours are now recognised in the WHO classification of CNS tumours and have been associated with a poor prognosis. Seven ZFTA-RELA fusion variants have been described: around two thirds of cases are fusion 1. No spontaneous genetically modified mouse models (GEMMS) have been described and current models require invasive intracranial injection (of transduced cells or RCAS-TVA system). Here we describe the first spontaneous GEMM of ZFTA-RELA fusion-driven ependymoma. Nestin-Flx-STOP-Flx-ZFTA-RELA (Fusion 1) open reading frames were targeted together with luciferase to the Rosa-26 locus. Breeding these mice with Nestin-CreERT2 or Blbp-Cre lines that drive recombination in neural progenitor cells resulted in forebrain tumours that could be tracked with bioluminescence imaging from P20. Tumours displayed NF-kB and L1CAM expression and ZFTA-RELA protein was detected using a novel in-house antibody. Tumours display expression of a known ZFTA-RELA fusion ependymoma transcriptomic signature. ZFTA-RELA tumours can be grown as neurospheres and passaged as allografts in nude mice and respond to irradiation. We provide the first spontaneous GEMM of this important group of ependymomas. Here we present our histologic and transcriptomic developmental timecourse of these tumours, their comparison to the human disease and further understanding of the lineage origins and potential novel targets.

JAK2V617F mutation drives vascular resident macrophages toward a pathogenic phenotype and promotes dissecting aortic aneurysm

JAK2V617F mutation is associated with an increased risk for athero-thrombotic cardiovascular disease, but its role in aortic disease development and complications remains unknown. In a cohort of patients with myeloproliferative neoplasm, JAK2V617F mutation was identified as an independent risk factor for dilation of both the ascending and descending thoracic aorta. Using single-cell RNA-seq, complementary genetically-modified mouse models, as well as pharmacological approaches, we found that JAK2V617F mutation was associated with a pathogenic pro-inflammatory phenotype of perivascular tissue-resident macrophages, which promoted deleterious aortic wall remodeling at early stages, and dissecting aneurysm through the recruitment of circulating monocytes at later stages. Finally, genetic manipulation of tissue-resident macrophages, or treatment with a Jak2 inhibitor, ruxolitinib, mitigated aortic wall inflammation and reduced aortic dilation and rupture. Overall, JAK2V617F mutation drives vascular resident macrophages toward a pathogenic phenotype and promotes dissecting aortic aneurysm.

Quaking but not parkin is the major tumor suppressor in 6q deleted region in glioblastoma.

Glioblastoma (GBM) is a high-grade, aggressive brain tumor with dismal median survival time of 15 months. Chromosome 6q (Ch6q) is a hotspot of genomic alterations, which is commonly deleted or hyper-methylated in GBM. Two neighboring genes in this region, QKI and PRKN have been appointed as tumor suppressors in GBM. While a genetically modified mouse model (GEMM) of GBM has been successfully generated with Qk deletion in the central nervous system (CNS), in vivo genetic evidence supporting the tumor suppressor function of Prkn has not been established. In the present study, we generated a mouse model with Prkn-null allele and conditional Trp53 and Pten deletions in the neural stem cells (NSCs) and compared the tumorigenicity of this model to our previous GBM model with Qk deletion within the same system. We find that Qk but not Prkn is the potent tumor suppressor in the frequently altered Ch6q region in GBM.

EPEN-25. A novel spontaneous model of ZFTA-RELA fusion ependymoma

Ependymomas driven by the ZFTA-RELA fusion account for >70% of all supratentorial ependymomas. These tumours are now recognised in the WHO classification of CNS tumours and have been associated with a poor prognosis. Seven ZFTA-RELA fusion variants have been described: around two thirds of cases are fusion 1. No spontaneous genetically modified mouse models (GEMMS) have been described and current models require invasive intracranial injection (of transduced cells or RCAS-TVA system). Here we describe the first spontaneous GEMM of ZFTA-RELA fusion-driven ependymoma. Nestin-Flx-STOP-Flx-ZFTA-RELA (Fusion 1) or E1alpha-Flx-STOP-Flx-ZFTA-RELA open reading frames were targeted together with luciferase to the Rosa-26 locus. Breeding these mice with Nestin-CreERT2 or Blbp-Cre lines that drive recombination in neural progenitor cells resulted in forebrain tumours that could be tracked with bioluminescence imaging from P20. Tumours displayed NF-kB and L1CAM expression and ZFTA-RELA protein was detected using a novel in-house antibody. Tumours display expression of a known ZFTA-RELA fusion ependymoma transcriptomic signature. ZFTA-RELA tumours can be grown as neurospheres and passaged as allografts in nude mice. We provide the first spontaneous GEMM of this important group of ependymomas. We are now characterising these tumours histologically and transcriptomically relative to the human disease and using these to understand the lineage origins of ependymoma and plan use of conventional and novel treatments.

The role of drug efflux and uptake transporters ABCB1 (P-gp), ABCG2 (BCRP) and OATP1A/1B and of CYP3A4 in the pharmacokinetics of the CDK inhibitor milciclib

The promising anticancer drug milciclib potently inhibits cyclin-dependent kinase (CDK) 2 and tropomyosin receptor kinase (TRK) A, and is currently in phase II clinical studies. To characterize factors affecting milciclib pharmacokinetics, we investigated whether milciclib is a substrate of the multidrug efflux and uptake transporters ABCB1 (P-gp), ABCG2 (BCRP), and OATP1A/1B, and the drug-metabolizing enzyme CYP3A, using genetically-modified mouse models and Madin-Darby Canine Kidney (MDCK-II) cells. In vitro, milciclib was transported by mAbcg2, and this was inhibited by the ABCG2 inhibitor Ko143. Upon oral administration of milciclib, its plasma exposure in Abcb1a/1b−/−, Abcg2−/−, and Abcb1a/1b;Abcg2−/− mice was similar to that found in wild-type mice. Milciclib showed good brain penetration even in wild-type mice (brain-to-plasma ratio of 1.2), but this was further increased by 5.2-fold when both Abcb1 and Abcg2 were ablated, and to a lesser extent in single Abcb1- or Abcg2-deficient mice. Oatp1a/1b deficiency had only a minor impact on the milciclib plasma AUC0-24h and Cmax. The milciclib AUC0-8h increased 1.9-fold in Cyp3a−/− mice but decreased only 1.3-fold upon overexpression of human CYP3A4. Thus, ABCB1 and ABCG2 cooperatively limit milciclib brain penetration. The low impact of OATP1 and CYP3A could be clinically favorable for milciclib, reducing the risks of unintended drug-drug interactions or interindividual variation in CYP3A4 activity.

Soluble CD5 and CD6: Lymphocytic Class I Scavenger Receptors as Immunotherapeutic Agents.

CD5 and CD6 are closely related signal-transducing class I scavenger receptors mainly expressed on lymphocytes. Both receptors are involved in the modulation of the activation and differentiation cell processes triggered by clonotypic antigen-specific receptors present on T and B cells (TCR and BCR, respectively). To serve such a relevant immunomodulatory function, the extracellular region of CD5 and CD6 interacts with soluble and/or cell-bound endogenous counterreceptors but also microbial-associated molecular patterns (MAMPs). Evidence from genetically-modified mouse models indicates that the absence or blockade of CD5- and CD6-mediated signals results in dysregulated immune responses, which may be deleterious or advantageous in some pathological conditions, such as infection, cancer or autoimmunity. Bench to bedside translation from transgenic data is constrained by ethical concerns which can be overcome by exogenous administration of soluble proteins acting as decoy receptors and leading to transient “functional knockdown”. This review gathers information currently available on the therapeutic efficacy of soluble CD5 and CD6 receptor infusion in different experimental models of disease. The existing proof-of-concept warrants the interest of soluble CD5 and CD6 as safe and efficient immunotherapeutic agents in diverse and relevant pathological conditions.

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity.

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a directin vivoCRISPRscreen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.

Wnt signaling and Loxl2 promote aggressive osteosarcoma

Osteosarcoma (OS) is the most frequent primary malignant bone tumor in urgent need of better therapies. Using genetically modified mouse models (GEMMs), we demonstrate that Wnt signaling promotes c-Fos-induced OS formation via the actions of the collagen-modifying enzyme Loxl2. c-Fos/AP-1 directly regulates the expression of the Wnt ligands Wnt7b and Wnt9a in OS cells through promoter binding, and Wnt7b and Wnt9a in turn promote Loxl2 expression in murine and human OS cells through the transcription factors Zeb1 and Zeb2. Concordantly, inhibition of Wnt ligand secretion by inactivating the Wnt-less (Wls) gene in osteoblasts in c-Fos GEMMs either early or in a therapeutic setting reduces Loxl2 expression and progression of OS. Wls-deficient osteosarcomas proliferate less, are less mineralized and are enriched in fibroblastic cells surrounded by collagen fibers. Importantly, Loxl2 inhibition using either the pan-Lox inhibitor BAPN or a specific inducible shRNA reduces OS cell proliferation in vitro and decreases tumor growth and lung colonization in murine and human orthotopic OS transplantation models. Finally, OS development is delayed in c-Fos GEMMs treated with BAPN or with specific Loxl2 blocking antibodies. Congruently, a strong correlation between c-FOS, LOXL2 and WNT7B/WNT9A expression is observed in human OS samples, and c-FOS/LOXL2 co-expression correlates with OS aggressiveness and decreased patient survival. Therefore, therapeutic targeting of Wnt and/or Loxl2 should be considered to potentiate the inadequate current treatments for pediatric, recurrent, and metastatic OS.

Behavioral tests assessing neuropsychiatric phenotypes in adolescent mice reveal strain- and sex-specific effects

In humans, infancy and adolescence are associated with major changes in synaptic functions and ongoing maturation of neural networks, which underlie the major behavioral changes during these periods. Among adult cases with neuropsychiatric disorders including autism spectrum disorder, schizophrenia, attention deficit hyperactivity, and bipolar disorders, 50% have developed behavioral symptoms and received a diagnosis before 15 years of age. However, most of the behavioral studies in mice modeling neuropsychiatric phenotypes are performed in adult animals, missing valuable phenotypic information related to the effect of synaptic maturation during development. Here, we explored which behavioral experiments assessing neuropsychiatric phenotypes can be performed during a specific window of development in adolescent male and female C57BL/6N, DBA/2, and FVB/N mice that are typically used as background strains for generating genetically-modified mouse models. The three wild-type strains were evaluated across anxiety, social behaviors, and cognitive functions in order to cover the main behavioral impairments that occur in neuropsychiatric disorders. During adolescence, the three strains displayed significant differences under certain behavioral paradigms. In addition, C57BL/6N and FVB/N, but not DBA/2 mice revealed some sex-related differences. Our results provide new insights into discrete behaviors during development and emphasize the crucial importance of the genetic background, sex, and experimental settings in the age-dependent regulation of different behaviors.

296 Recombination efficiency and expression of fibroblast-specific Cre drivers in mouse skin

Cell-type specific genetic recombination is a powerful strategy for advancing understanding of cellular function in genetically-modified mouse models. This approach relies on cell-type specific gene promoters to drive genetic recombination in target cells. Although the utility of many cell-type specific promoters has been well characterized, thorough characterization of fibroblast-specific promoters is lacking. Therefore, we employed the mTmG two-color fluorescent reporter system to examine the genetic recombination efficiency and expression patterns of three fibroblast-specific promoters in mouse skin. mTmG mice express the red fluorescent protein tdTomato (mT) constitutively and ubiquitously. Genetic recombination catalyzed by Cre recombinase silences mT expression and induces expression of enhanced green fluorescent protein (mG). Thus, cells that express Cre recombinase appear green, while all other cells appear red. We generated mTmG mice that express Cre recombinase under the control of promoter elements in three fibroblast-specific genes 1) fibroblast specific protein-1 (FSP-1), 2) type Iα2 collagen (COL1A2), or 3) platelet-derived growth factor receptor-α (PDGFRα). Dorsal skin from mTmG/Cre positive and control mice were analyzed by immunofluorescence microscopy. Surprisingly, mTmG/FSP-1 Cre mice exhibited recombination largely in keratinocytes throughout all the layers of the epidermis and follicular epithelium. Recombination was also observed in scattered fibroblast-like cells in the dermis. mTmG/COL1A2 Cre mice displayed recombination that was specific for dermal cells with fibroblast-like morphology throughout the interstitial dermis and in dermal papillae. mTmG/PDGFRα Cre mice displayed recombination that was similar to that of COL1A2-driven recombination, although the number of positive dermal cells and the intensity of mG expression was approximately five-fold greater. These data provide a foundation for the use of Cre drivers to study fibroblast gene expression in genetically-modified mouse models.

Supervised learning to quantify amyloidosis in whole brains of an Alzheimer's disease mouse model acquired with optical projection tomography.

Alzheimer's disease (AD) is characterized by amyloidosis of brain tissues. This phenomenon is studied with genetically-modified mouse models. We propose a method to quantify amyloidosis in whole 5xFAD mouse brains, a model of AD. We use optical projection tomography (OPT) and a random forest voxel classifier to segment and measure amyloid plaques. We validate our method in a preliminary cross-sectional study, where we measure 6136 ± 1637, 8477 ± 3438, and 17267 ± 4241 plaques (AVG ± SD) at 11, 17, and 31 weeks. Overall, this method can be used in the evaluation of new treatments against AD.

BMAL1 Links Bevacizumab Resistance in Colorectal Cancer to Circadian Rhythm and Heme Receptor REVERBA

Heme sensor Nuclear Receptor Subfamily 1 Group D Member 1 (NR1D1/REVERBA) and its target gene Aryl Hydrocarbon Receptor Nuclear Translocator-Like (ARNTL/BMAL1) regulate circadian rhythm and angiogenesis. Given their involvement in the interplay between clock disruptions (e.g. by mutations or sleep deprival), cancer risk and vascular biology, we hypothesized that these transcription factors contribute to resistance against anti-angiogenic therapy with vascular endothelial growth factor (VEGFA) neutralizing antibody (bevacizumab, abbrev. Beva) in colorectal cancer (CRC). REVERBA bound to a predicted -672 bp Retinoic Acid Receptor-Related Orphan Receptor Alpha (RORA)-responsive element (RORE) adjacent to a BMAL1 DNA-binding motif (E-box) in the proximal promoter of the human VEGFA gene. REVERBA, indirectly and together with BMAL1, increased VEGFA mRNA and protein expression. Conversely, REVERBA siRNA and REVERBA-antagonist hemin decreased VEGFA synthesis. Beva treatment inhibited CRC growth in a C57BL/6J Apc min/+ genetically modified mouse model (GEMM) and BALB/c nu/nu mouse HCT116 xenografts, without attenuating BMAL1 levels or microvessel density (MVD). In CRC patient tumors (n=74), high BMAL1 protein expression was associated with clinical non-response to combination chemotherapy with Beva (*p=0.0061) and reduced progression-free survival (PFS) [*p=0.0223, Hazard Ratio (HR)=1.69]. In addition, single nucleotide polymorphisms (SNPs) in the BMAL1 (ARNTL) gene correlated with shorter overall survival (OS) [rs11022780, *p=0.014, HR=1.61] and PFS (rs7396943, rs7938307, rs2279287) in patients undergoing combination chemotherapy with Beva. Thus, BMAL1 is associated with Beva resistance in CRC. Since the REVERBA-BMAL1-VEGFA axis can be pharmacologically targeted, interference with this signaling pathway could circumvent resistance to anti-angiogenic therapy in CRC. Funding Statement: This study was supported by grants to EB from the Deutsche Krebshilfe (#108287 and #111086). FE received a fellowship from the “Studienstiftung des deutschen Volkes”. JB was supported by the Translational Physician Scientist (TraPS) Program of the Medical Faculty Mannheim of the Heidelberg University. WW was awarded a fellowship from the Chinese Scholarship Council (#201408080116). ME was supported by the State of Baden-Wurttemberg for “Center of Geriatric Biology and Oncology (ZOBEL) – Perspektivforderung” and “Biology of Frailty - Sonderlinie Medizin”. ATB and ME received funds from the European Commission Seventh Framework Programme (Contract No. 278981 [ANGIOPREDICT]) and are supported by the European Commission Horizon 2020 Programme (Contract No. 754923 [COLOSSUS]). ATB is further supported by Science Foundation Ireland under grant 13/CDA/2183 ”Coloforetell”. HJL was partly funded by the National Cancer Institute (grant number P30CA014089), the Gloria Borges WunderGlo Foundation -The Wunder Project, the Dhont Family Foundation, the San Pedro Peninsula Cancer Guild, the Daniel Butler Research Fund, and the Call to Cure Research Fund Declaration of Interests: The authors declare no conflicts of interest. The reagent mBeva was obtained on the basis of a material transfer agreement (MTA#10082012) from Roche Diagnostics GmbH (Penzberg, Germany). HJL has received clinical trial financial support from Merck Serono and Roche and honoraria for advisory board membership and lectures from Bayer, Boehringer Ingelheim, Genentech, Merck Serono and Roche Ethics Approval Statement: Xenograft studies conformed to the 2010/63/EU guidelines and were approved by the Dept. of Health and Children, Dublin, Ireland (B100/3654) and University College Dublin Animal Research Ethics Committee (P12-27). Therapy studies were conducted in accordance with ethical guidelines (Declaration of Helsinki) and approved by the local ethics committees for each participating site. All patients provided written informed consent for the analysis of molecular correlates.

Mouse Models of Melanoma Bone Metastasis.

Melanomas are aggressive cancers of the skin with high metastatic capacity. Mouse models are necessary to delineate the mechanisms of cancer metastasis and xenograft models can also allow examining the role of the host using different genetically-modified mouse models. In this chapter, I provide a detailed protocol for the preparation and inoculation of tumor cells intra-cardially and intra-tibially to achieve bone metastasis.

Abstract 4778: Assessing efficacy of neratinib in HER2-driven lung cancer

Abstract HER2 mutations have been classified as oncogenic drivers that are present in 2-4% of lung adenocarcinomas. However, due to limited preclinical studies and clinical trials, there is still no available standard of care for this group of lung cancer patients. To fulfill the clinical need for targeting HER2 mutations in lung cancer patients, we performed a comprehensive preclinical study to evaluate the efficacy of neratinib, an irreversible small-molecule pan-HER tyrosine kinase inhibitor that potently binds and inhibits EGFR, HER2, and HER4. We used the previously described genetically modified mouse model (GEMM) of lung adenocarcinomas driven by the major mutant form of human HER2 exon 20 insertions (A775_G776insYVMA) for testing neratinib either as a single agent or in combination with other therapies including trastuzumab in vivo. Treatment efficacy was evaluated based on both monitoring tumor volume changes and survival. Magnetic resonance imaging (MRI) was used for quantifying tumor burden. Tolerability and the overall health of animals were evaluated based on body weight change. We found that neratinib treatment alone provided a modest yet significant response in inhibiting HER2 mutant lung tumors in this model. Pharmacodynamic studies showed that neratinib effectively abolished HER2 phosphorylation and inhibited major downstream signaling targets such as pAKT and pERK. Moreover, combination therapy with neratinib and trastuzumab showed a greater treatment effect by reducing tumor burden with significantly longer progression-free survival and overall survival compared to single agents tested. In all neratinib-based regimens, body weight of treated animals remained stable. On this basis, our study provides a strong rationale to test the combination of neratinib with trastuzumab for the treatment of HER2 mutant NSCLC patients. Citation Format: Shuai Li, Shengwu Liu, Ting Chen, Jiehui Deng, Min Wu, Mari Kuraguchi, Cam Anh Tran, Paul T. Kirschmeier, Francesca Avogadri-Connors, Richard E. Cutler, Alshad S. Lalani, Kwok-Kin Wong. Assessing efficacy of neratinib in HER2-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4778.

Abstract LB-262: Design, synthesis, and efficacy of MYC inhibitors in mouse pancreatic cancer

Abstract Therapeutic inhibition of the MYC oncoprotein action for cancer treatment is a challenging aim and various anti-MYC approaches, including the development of small-molecule candidate drugs, are being actively pursued. Using genetically-modified mouse models of cancer, we have demonstrated that the oncogenicity of mutant KRAS causally depends on unperturbed MYC activity. On the basis of this observation, we performed preclinical studies and treated successfully mouse pancreatic, breast and other cancers, and also xenografts of human pancreatic cancer cell lines, by using Mycro3, an orally-bioavailable small-molecule inhibitor of MYC-MAX dimerization. We observed that, in comparison with vehicle-treated controls, tumor growth in Mycro3-treated mice was drastically attenuated.1 To optimize Mycro3, we have used drug design, organic synthesis, in vitro assays, pharmacokinetic profiling, and in vivo efficacy studies. These efforts led to the identification of novel derivatives, one of which (Amy22) exhibits a vastly improved therapeutic window against MYC (>300-fold over Mycro3). It has also superior physicochemical and pharmacokinetic properties (the peak blood concentration is >6-fold better over Mycro3, while the compound bioavailability is maintained for more than 12 hours). The designed candidate anti-Myc compounds were synthesized by coupling properly substituted pyrazolo[1,5-a]pyrimidines with various amines, and then evaluated in vitro using the TGR-1 (Myc+/+) and H015.19 (Myc-/-) sibling cell lines. The in vivo efficacy of Amy22 has been tested successfully in preclinical studies using advantageous mouse models of MYC overexpressing tumors that we have developed, which enable rapid evaluations. Moreover, patient-derived xenografts (PDXs) are being used, while combinatorial therapeutic regimes using anti-Myc treatment with drugs inhibiting other collaborating tumorigenic pathways have been evaluated.2 ZC has been co-funded by the European Commission under the H2020 Research Infrastructures Contract No. 675121 (project VI-SEEM). 1D. Stellas, M. Szabolcs, S. Koul, Z. Li, A. Polyzos, C. Anagnostopoulos, Z. Cournia, C. Tamvakopoulos, A. Klinakis & A. Efstratiadis (2014) Therapeutic Effects of an anti-Myc Drug on Mouse Pancreatic Cancer, J Natl Cancer Inst, 106(12). pii: dju320 2D. Stellas, Z. Cournia, C. Tamvakopoulos, A. Klinakis & A. Efstratiadis. “Novel Compounds for use in treating or preventing cancerous diseases”. International Patent Application number PCT/EP2016/066340 Citation Format: Veroniki P. Vidali, Zoe Cournia, Adriana Papadimitropoulou, Dimitrios Stellas, Apostolos Klinakis, Elias A. Couladouros, Constantin Tamvakopoulos, Argiris Efstratiadis. Design, synthesis, and efficacy of MYC inhibitors in mouse pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-262.

Targeting HER2 Aberrations in Non-Small Cell Lung Cancer with Osimertinib.

Purpose:HER2 (or ERBB2) aberrations, including both amplification and mutations, have been classified as oncogenic drivers that contribute to 2% to 6% of lung adenocarcinomas. HER2 amplification is also an important mechanism for acquired resistance to EGFR tyrosine kinase inhibitors (TKI). However, due to limited preclinical studies and clinical trials, currently there is still no available standard of care for lung cancer patients with HER2 aberrations. To fulfill the clinical need for targeting HER2 in patients with non-small cell lung cancer (NSCLC), we performed a comprehensive preclinical study to evaluate the efficacy of a third-generation TKI, osimertinib (AZD9291).Experimental Design: Three genetically modified mouse models (GEMM) mimicking individual HER2 alterations in NSCLC were generated, and osimertinib was tested for its efficacy against these HER2 aberrations in vivoResults: Osimertinib treatment showed robust efficacy in HER2wt overexpression and EGFR del19/HER2 models, but not in HER2 exon 20 insertion tumors. Interestingly, we further identified that combined treatment with osimertinib and the BET inhibitor JQ1 significantly increased the response rate in HER2-mutant NSCLC, whereas JQ1 single treatment did not show efficacy.Conclusions: Overall, our data indicated robust antitumor efficacy of osimertinib against multiple HER2 aberrations in lung cancer, either as a single agent or in combination with JQ1. Our study provides a strong rationale for future clinical trials using osimertinib either alone or in combination with epigenetic drugs to target aberrant HER2 in patients with NSCLC. Clin Cancer Res; 24(11); 2594-604. ©2018 AACRSee related commentary by Cappuzzo and Landi, p. 2470.