Spontaneous and Engineered Large Animal Models of Neurofibromatosis Type 1.

Animal models are crucial to understanding human disease biology and developing new therapies. By far the most common animal used to investigate prevailing questions about human disease is the mouse. Mouse models are powerful tools for research as their small size, limited lifespan, and defined genetic background allow researchers to easily manipulate their genome and maintain large numbers of animals in general laboratory spaces. However, it is precisely these attributes that make them so different from humans and explains, in part, why these models do not accurately predict drug responses in human patients. This is particularly true of the neurofibromatoses (NFs), a group of genetic diseases that predispose individuals to tumors of the nervous system, the most common of which is Neurofibromatosis type 1 (NF1). Despite years of research, there are still many unanswered questions and few effective treatments for NF1. Genetically engineered mice have drastically improved our understanding of many aspects of NF1, but they do not exemplify the overall complexity of the disease and some findings do not translate well to humans due to differences in body size and physiology. Moreover, NF1 mouse models are heavily reliant on the Cre-Lox system, which does not accurately reflect the molecular mechanism of spontaneous loss of heterozygosity that accompanies human tumor development. Spontaneous and genetically engineered large animal models may provide a valuable supplement to rodent studies for NF1. Naturally occurring comparative models of disease are an attractive prospect because they occur on heterogeneous genetic backgrounds and are due to spontaneous rather than engineered mutations. The use of animals with naturally occurring disease has been effective for studying osteosarcoma, lymphoma, and diabetes. Spontaneous NF-like symptoms including neurofibromas and malignant peripheral nerve sheath tumors (MPNST) have been documented in several large animal species and share biological and clinical similarities with human NF1. These animals could provide additional insight into the complex biology of NF1 and potentially provide a platform for pre-clinical trials. Additionally, genetically engineered porcine models of NF1 have recently been developed and display a variety of clinical features similar to those seen in NF1 patients. Their large size and relatively long lifespan allow for longitudinal imaging studies and evaluation of innovative surgical techniques using human equipment. Greater genetic, anatomic, and physiologic similarities to humans enable the engineering of precise disease alleles found in human patients and make them ideal for preclinical pharmacokinetic and pharmacodynamic studies of small molecule, cellular, and gene therapies prior to clinical trials in patients. Comparative genomic studies between humans and animals with naturally occurring disease, as well as preclinical studies in large animal disease models, may help identify new targets for therapeutic intervention and expedite the translation of new therapies. In this review, we discuss new genetically engineered large animal models of NF1 and cases of spontaneous NF-like manifestations in large animals, with a special emphasis on how these comparative models could act as a crucial translational intermediary between specialized murine models and NF1 patients.

Combining nonsense mutation suppression therapy with nonsense-mediated decay inhibition in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) results from germline mutations in the tumor-suppressor gene NF1 and predisposes patients to developing nervous system tumors. Twenty percent of NF1 patients harbor nonsense mutations resulting in premature termination codons (PTCs). Nonsense suppression therapies can facilitate ribosomal readthrough of PTCs to restore full-length protein, but their potential in NF1 is underexplored. We developed a minipig model of NF1 carrying a PTC to test whether nonsense suppression could restore expression of the NF1-encoded protein neurofibromin in vitro and in vivo. Nonsense suppression did not reliably increase neurofibromin in primary NF1-/- Schwann cells isolated from minipig neurofibromas but could reduce phosphorylated ERK. Gentamicin in vivo produced a similar plasma pharmacokinetic profile to humans and was detectable in clinically relevant tissues, including cerebral cortex, sciatic nerve, optic nerve, and skin. In gentamicin-treated animals, increased neurofibromin expression was seen in the optic nerve. Nonsense-mediated decay (NMD) causes degradation of transcripts with PTCs, which could impede nonsense suppression therapies. Nonsense suppression in combination with NMD inhibition restored neurofibromin protein expression in primary NF1-/- Schwann cells isolated from minipig neurofibromas. Thus, the effectiveness of nonsense suppression therapies can be improved in NF1 by the concurrent use of NMD inhibitors.

Ex vivo to in vivo model of malignant peripheral nerve sheath tumors for precision oncology.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that often develop in patients with neurofibromatosis type 1 (NF1). To address the critical need for novel therapeutics in MPNST, we aimed to establish an ex vivo 3D platform that accurately captured the genomic diversity of MPNST and could be utilized in a medium-throughput manner for drug screening studies to be validated in vivo using patient-derived xenografts (PDX). METHODS: Genomic analysis was performed on all PDX-tumor pairs. Selected PDX were harvested for assembly into 3D microtissues. Based on prior work in our labs, we evaluated drugs (trabectedin, olaparib, and mirdametinib) ex vivo and in vivo. For 3D microtissue studies, cell viability was the endpoint as assessed by Zeiss Axio Observer. For PDX drug studies, tumor volume was measured twice weekly. Bulk RNA sequencing was performed to identify pathways enriched in cells. RESULTS: We developed 13 NF1-associated MPNST-PDX and identified mutations or structural abnormalities in NF1 (100%), SUZ12 (85%), EED (15%), TP53 (15%), CDKN2A (85%), and chromosome 8 gain (77%). We successfully assembled PDX into 3D microtissues, categorized as robust (>90% viability at 48 h), good (>50%), or unusable (<50%). We evaluated drug response to "robust" or "good" microtissues, namely MN-2, JH-2-002, JH-2-079-c, and WU-225. Drug response ex vivo predicted drug response in vivo, and enhanced drug effects were observed in select models. CONCLUSIONS: These data support the successful establishment of a novel 3D platform for drug discovery and MPNST biology exploration in a system representative of the human condition.

Selumetinib normalizes Ras/MAPK signaling in clinically relevant neurofibromatosis type 1 minipig tissues in vivo.

BACKGROUND: The MEK1/2 inhibitor selumetinib was recently approved for neurofibromatosis type 1 (NF1)-associated plexiform neurofibromas, but outcomes could be improved and its pharmacodynamic evaluation in other relevant tissues is limited. The aim of this study was to assess selumetinib tissue pharmacokinetics (PK) and pharmacodynamics (PD) using a minipig model of NF1. METHODS: WT (n = 8) and NF1 (n = 8) minipigs received a single oral dose of 7.3 mg/kg selumetinib. Peripheral blood mononuclear cells (PBMCs), cerebral cortex, optic nerve, sciatic nerve, and skin were collected for PK analysis and PD analysis of extracellular regulated kinase phosphorylation (p-ERK) inhibition and transcript biomarkers (DUSP6 & FOS). RESULTS: Key selumetinib PK parameters aligned with those observed in human patients. Selumetinib concentrations were higher in CNS tissues from NF1 compared to WT animals. Inhibition of ERK phosphorylation was achieved in PBMCs (mean 60% reduction), skin (95%), and sciatic nerve (64%) from all minipigs, whereas inhibition of ERK phosphorylation in cerebral cortex was detected only in NF1 animals (71%). Basal p-ERK levels were significantly higher in NF1 minipig optic nerve compared to WT and were reduced to WT levels (60%) with selumetinib. Modulation of transcript biomarkers was observed in all tissues. CONCLUSIONS: Selumetinib reduces MAPK signaling in tissues clinically relevant to NF1, effectively normalizing p-ERK to WT levels in optic nerve but resulting in abnormally low levels of p-ERK in the skin. These results suggest that selumetinib exerts activity in NF1-associated CNS tumors by normalizing Ras/MAPK signaling and may explain common MEK inhibitor-associated dermatologic toxicities.

Intestinal CD8αα IELs derived from two distinct thymic precursors have staggered ontogeny.

CD8αα intraepithelial lymphocytes (IELs) are abundant T cells that protect the gut epithelium. Their thymic precursors (IELps) include PD-1+ type A and Tbet+ type B populations, which differ in their antigen-receptor specificities. To better understand CD8αα IEL ontogeny, we performed "time-stamp" fate mapping experiments and observed that it seeds the intestine predominantly during a narrow time window in early life. Adoptively transferred IELps parked better in the intestines of young mice than in adults. In young mice, both type A and type B IELps had an S1PR1+ and α4ß7+ emigration- and mucosal-homing competent phenotype, while this was restricted to type A IELps in adults. Only CD8αα IELs established in early life were enriched in cells bearing type B IELp TCR usage. Together, our results suggest that the young intestine facilitates CD8αα IEL establishment and that early IELs are distinct from IELs established after this initial wave. These data provide novel insight into the ontogeny of CD8αα IELs.

PLX3397 treatment inhibits constitutive CSF1R-induced oncogenic ERK signaling, reduces tumor growth, and metastatic burden in osteosarcoma.

Osteosarcoma (OSA) is a heterogeneous and aggressive solid tumor of the bone. We recently identified the colony stimulating factor 1 receptor (Csf1r) gene as a novel driver of osteosarcomagenesis in mice using the Sleeping Beauty (SB) transposon mutagenesis system. Here, we report that a CSF1R-CSF1 autocrine/paracrine signaling mechanism is constitutively activated in a subset of human OSA cases and is critical for promoting tumor growth and contributes to metastasis. We examined CSF1R and CSF1 expression in OSAs. We utilized gain-of-function and loss-of-function studies (GOF/LOF) to evaluate properties of cellular transformation, downstream signaling, and mechanisms of CSF1R-CSF1 action. Genetic perturbation of CSF1R in immortalized osteoblasts and human OSA cell lines significantly altered oncogenic properties, which were dependent on the CSF1R-CSF1 autocrine/paracrine signaling. These functional alterations were associated with changes in the known CSF1R downstream ERK effector pathway and mitotic cell cycle arrest. We evaluated the recently FDA-approved CSF1R inhibitor Pexidartinib (PLX3397) in OSA cell lines in vitro and in vivo in cell line and patient-derived xenografts. Pharmacological inhibition of CSF1R signaling recapitulated the in vitro genetic alterations. Moreover, in orthotopic OSA cell line and subcutaneous patient-derived xenograft (PDX)-injected mouse models, PLX3397 treatment significantly inhibited local OSA tumor growth and lessened metastatic burden. In summary, CSF1R is utilized by OSA cells to promote tumorigenesis and may represent a new molecular target for therapy.