Targeted inactivation of Mdm2 RING finger E3 ubiquitin ligase activity in the mouse reveals mechanistic insights into p53 regulation.

It is believed that Mdm2 suppresses p53 in two ways: transcriptional inhibition by direct binding, and degradation via its E3 ligase activity. To study these functions physiologically, we generated mice bearing a single-residue substitution (C462A) abolishing the E3 function without affecting p53 binding. Unexpectedly, homozygous mutant mice died before E7.5, and deletion of p53 rescued the lethality. Furthermore, reintroducing a switchable p53 by crossing with p53ER(TAM) mice surprisingly demonstrated that the mutant Mdm2(C462A) was rapidly degraded in a manner indistinguishable from that of the wild-type Mdm2. Hence, our data indicate that (1) the Mdm2-p53 physical interaction, without Mdm2-mediated p53 ubiquitination, cannot control p53 activity sufficiently to allow early mouse embryonic development, and (2) Mdm2's E3 function is not required for Mdm2 degradation.

The Collaborative Cross as a resource for modeling human disease: CC011/Unc, a new mouse model for spontaneous colitis.

Inflammatory bowel disease (IBD) is an immune-mediated condition driven by improper responses to intestinal microflora in the context of environmental and genetic background. GWAS in humans have identified many loci associated with IBD, but animal models are valuable for dissecting the underlying molecular mechanisms, characterizing environmental and genetic contributions and developing treatments. Mouse models rely on interventions such as chemical treatment or introduction of an infectious agent to induce disease. Here, we describe a new model for IBD in which the disease develops spontaneously in 20-week-old mice in the absence of known murine pathogens. The model is part of the Collaborative Cross and came to our attention due to a high incidence of rectal prolapse in an incompletely inbred line. Necropsies revealed a profound proliferative colitis with variable degrees of ulceration and vasculitis, splenomegaly and enlarged mesenteric lymph nodes with no discernible anomalies of other organ systems. Phenotypic characterization of the CC011/Unc mice with homozygosity ranging from 94.1 to 99.8% suggested that the trait was fixed and acted recessively in crosses to the colitis-resistant C57BL/6J inbred strain. Using a QTL approach, we identified four loci, Ccc1, Ccc2, Ccc3 and Ccc4 on chromosomes 12, 14, 1 and 8 that collectively explain 27.7% of the phenotypic variation. Surprisingly, we also found that minute levels of residual heterozygosity in CC011/Unc have significant impact on the phenotype. This work demonstrates the utility of the CC as a source of models of human disease that arises through new combinations of alleles at susceptibility loci.

Enhancing CDK4/6 inhibitor therapy for medulloblastoma using nanoparticle delivery and scRNA-seq-guided combination with sapanisertib.

The therapeutic potential of CDK4/6 inhibitors for brain tumors has been limited by recurrence. To address recurrence, we tested a nanoparticle formulation of CDK4/6 inhibitor palbociclib (POx-Palbo) in mice genetically-engineered to develop SHH-driven medulloblastoma, alone or in combination with specific agents suggested by our analysis. Nanoparticle encapsulation reduced palbociclib toxicity, enabled parenteral administration, improved CNS pharmacokinetics, and extended mouse survival, but recurrence persisted. scRNA-seq identified up-regulation of glutamate transporter Slc1a2 and down-regulation of diverse ribosomal genes in proliferating medulloblastoma cells in POx-Palbo-treated mice, suggesting mTORC1 signaling suppression, subsequently confirmed by decreased 4EBP1 phosphorylation. Combining POx-Palbo with the mTORC1 inhibitor sapanisertib produced mutually enhancing effects and prolonged mouse survival compared to either agent alone, contrasting markedly with other tested drug combinations. Our data show the potential of nanoparticle formulation and scRNA-seq analysis of resistance to improve brain tumor treatment and identify POx-Palbo + Sapanisertib as effective combinatorial therapy for SHH medulloblastoma.

The Scurfy mutation of FoxP3 in the thymus stroma leads to defective thymopoiesis.

The Scurfy mutation of the FoxP3 gene (FoxP3(sf)) in the mouse and analogous mutations in human result in lethal autoimmunity. The mutation of FoxP3 in the hematopoietic cells impairs the development of regulatory T cells. In addition, development of the Scurfy disease also may require mutation of the gene in nonhematopoietic cells. The T cell-extrinsic function of FoxP3 has not been characterized. Here we show that the FoxP3(sf) mutation leads to defective thymopoiesis, which is caused by inactivation of FoxP3 in the thymic stromal cells. FoxP3 mutation also results in overexpression of ErbB2 in the thymic stroma, which may be involved in defective thymopoiesis. Our data reveal a novel T cell-extrinsic function of FoxP3. In combination, the T cell-intrinsic and -extrinsic defects provide plausible explanation for the severity of the autoimmune diseases in the scurfy mice and in patients who have immunodysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome.

Development of outbred CD1 mouse colonies with distinct standardized gut microbiota profiles for use in complex microbiota targeted studies.

Studies indicate that the gut microbiota (GM) can significantly influence both local and systemic host physiologic processes. With rising concern for optimization of experimental reproducibility and translatability, it is essential to consider the GM in study design. However, GM profiles can vary between rodent producers making consistency between models challenging. To circumvent this, we developed outbred CD1 mouse colonies with stable, complex GM profiles that can be used as donors for a variety of GM transfer techniques including rederivation, co-housing, cross-foster, and fecal microbiota transfer (FMT). CD1 embryos were surgically transferred into CD1 or C57BL/6 surrogate dams that varied by GM composition and complexity to establish four separate mouse colonies harboring GM profiles representative of contemporary mouse producers. Using targeted 16S rRNA amplicon sequencing, subsequent female offspring were found to have similar GM profiles to surrogate dams. Furthermore, breeding colonies of CD1 mice with distinct GM profiles were maintained for nine generations, demonstrating GM stability within these colonies. To confirm GM stability, we shipped cohorts of these four colonies to collaborating institutions and found no significant variation in GM composition. These mice are an invaluable experimental resource that can be used to investigate GM effects on mouse model phenotype.

Haploinsufficiency of p18(INK4c) sensitizes mice to carcinogen-induced tumorigenesis.

The INK4 family of cyclin-dependent kinase (CDK) inhibitors negatively regulates cyclin D-dependent CDK4 and CDK6 and thereby retains the growth-suppressive function of Rb family proteins. Mutations in the CDK4 gene conferring INK4 resistance are associated with familial and sporadic melanoma in humans and result in a wide spectrum of tumors in mice. Whereas loss of function of other INK4 genes in mice leads to little or no tumor development, targeted deletion of p18(INK4c) causes spontaneous pituitary tumors and lymphoma late in life. Here we show that treatment of p18 null and heterozygous mice with a chemical carcinogen resulted in tumor development at an accelerated rate. The remaining wild-type allele of p18 was neither mutated nor silenced in tumors derived from heterozygotes. Hence, p18 is a haploinsufficient tumor suppressor in mice.

Epiregulin is not essential for development of intestinal tumors but is required for protection from intestinal damage.

Epiregulin, an epidermal growth factor family member, acts as a local signal mediator and shows dual biological activity, stimulating the proliferation of fibroblasts, hepatocytes, smooth muscle cells, and keratinocytes while inhibiting the growth of several tumor-derived epithelial cell lines. The epiregulin gene (Ereg) is located on mouse chromosome 5 adjacent to three other epidermal growth factor family members, epigen, amphiregulin, and betacellulin. Gene targeting was used to insert a lacZ reporter into the mouse Ereg locus and to ablate its function. Although epiregulin is broadly expressed and regulated both spatially and temporally, Ereg null mice show no overt developmental defects, reproductive abnormalities, or altered liver regeneration. Additionally, in contrast to previous hypotheses, Ereg deficiency does not alter intestinal cancer susceptibility, as assayed in the ApcMin model, despite showing robust expression in developing tumors. However, Ereg null mice are highly susceptible to cancer-predisposing intestinal damage caused by oral administration of dextran sulfate sodium.

Azoxymethane is a genetic background-dependent colorectal tumor initiator and promoter in mice: effects of dose, route, and diet.

The azoxymethane (AOM) model has been widely used to investigate the pathology and genetics of colorectal cancer in rodents. However, there has been wide variation in treatment regimes, making it difficult to compare across studies. Consequently, standardizing AOM treatment and identifying sources of experimental variation would allow better comparisons across studies. In order to establish an optimal dosing regime for detecting experiment-dependent differences in tumorigenesis, we performed a dose curve analysis using AKR/J, SWR/J, and A/J mouse strains previously reported to vary widely in susceptibility to AOM. Although intraperitoneal or subcutaneous administration, but not in utero exposure, resulted in similar levels of tumor induction, significant dose- and strain-dependent effects of AOM were observed. No sex-dependent differences were observed. Increasing the number of treatments uncovered a significant strain-dependent effect on tumor promotion, independent of susceptibility to tumor initiation. Similarly, we used C57BL/6J and DBA/2J intercrosses to demonstrate that small diet modifications can significantly alter AOM-induced tumorigenesis in a background-dependent manner. These results provide experimental support for a standardized AOM treatment and for the importance of controlling both genetic and non-genetic factors when using this model.

Metastatic uterine adenocarcinoma in an 8-year-old gilt.

An 8-y-old gilt was evaluated after the onset of hemorrhagic perineal discharge. Uterine adenocarcinoma with metastases to the lungs and regional lymph nodes was diagnosed at necropsy. Tumor cells lacked expression of estrogen receptor alpha and progesterone receptor. This case represents the first reported uterine adenocarcinoma in a research pig and the first swine uterine neoplasia in which steroid hormone receptor expression was evaluated.

Biodegradation of Inion fast-absorbing biodegradable plates and screws.

Biodegradable plates and screws are recommended for use in surgery of the craniofacial skeleton of children. To be effective and not interfere with growth of the child's skull, the plates must biodegrade sufficiently to release the holding power of the plate and screw within 1 year. It is also essential that excessive foreign body reaction and cyst formation does not occur when the plates and screws biodegrade. The purpose of this experimental study was to evaluate the rate of biodegradation of Inion CPS Baby biodegradable plates and screws under different clinical circumstances in the rabbit craniofacial skeleton and evaluate their efficacy for use in pediatric craniofacial surgery. Foreign body reaction would be evaluated. Inion baby plates and screws were tested in a rabbit model. Plates were applied to the frontal bone, over a bony defect of the parietal bone, to a nasal bone fracture, and inserted in the subcutaneous space over the occipital bone in thirty 6-week-old rabbits. Six rabbits were euthanized at 9, 12, 15, and 18 months' postoperative time point and examined for residual plates and screws. Bone from each surgical site was excised, fixed by immersion in 10% neutral-buffered formalin, decalcified in Immunocal solution, and examined by 7-microm paraffin sections stained with hematoxylin and eosin. At 9 months, the plates and screws had effectively biodegraded and no longer had holding power on the bones. Fragmentation of the implant material was noted. Residual implant material was still present on gross and histologic examination in rabbits at 9, 12, 15, and 18 months. Residue of a screw was still palpable in 1 rabbit at 18 months. There was no evidence of cyst formation in any of the examined specimens. Macrophages and giant cells were present in most of the specimens at 9, 12, 15, and 18 months. Findings from the current study revealed a relative short resorption time (9 mo) and normal inflammatory sequelae in an adult rabbit model. These findings suggest that these plates may be used safely in fixing the pediatric craniofacial skeleton.

Overexpression of Eg5 causes genomic instability and tumor formation in mice.

Proper chromosome segregation in eukaryotes is driven by a complex superstructure called the mitotic spindle. Assembly, maintenance, and function of the spindle depend on centrosome migration, organization of microtubule arrays, and force generation by microtubule motors. Spindle pole migration and elongation are controlled by the unique balance of forces generated by antagonistic molecular motors that act upon microtubules of the mitotic spindle. Defects in components of this complex structure have been shown to lead to chromosome missegregation and genomic instability. Here, we show that overexpression of Eg5, a member of the Bim-C class of kinesin-related proteins, leads to disruption of normal spindle development, as we observe both monopolar and multipolar spindles in Eg5 transgenic mice. Our findings show that perturbation of the mitotic spindle leads to chromosomal missegregation and the accumulation of tetraploid cells. Aging of these mice revealed a higher incidence of tumor formation with a mixed array of tumor types appearing in mice ages 3 to 30 months with the mean age of 20 months. Analysis of the tumors revealed widespread aneuploidy and genetic instability, both hallmarks of nearly all solid tumors. Together with previous findings, our results indicate that Eg5 overexpression disrupts the unique balance of forces associated with normal spindle assembly and function, and thereby leads to the development of spindle defects, genetic instability, and tumors.

Foxp3 controls autoreactive T cell activation through transcriptional regulation of early growth response genes and E3 ubiquitin ligase genes, independently of thymic selection.

To elucidate the mechanisms of autoreactive T cell activation and expansion, we used endogenous viral superantigens (VSAg)-reactive T cells as a model of self-antigens in two strains of Foxp3-mutant mice. These two strains, together with wild-type mice, provided us with an advantage to simultaneously study the positively and negatively selected as well as rescued autoreactive T cells. We show here that while both VSAg-reactive and non-VSAg-reactive T cells are equally activated in Foxp3-mutant mice, only the VSAg-reactive T cells are preferentially expanded independently of their selected states in the thymus. The T cell activation appears to be controlled by Foxp3 through transcriptional regulation of early growth response (Egr) genes Egr-2 and Egr-3, and E3 ubiquitin (Ub) ligase genes Cblb, Itch and GRAIL, subsequently affecting degradation of two key signaling proteins, PLCgamma1 and PKC-theta. Physiologically, the positively, but not negatively selected VSAg-reactive T cells are spontaneously activated without significant expansion. The results suggest that autoreactive T cell activation is controlled by Foxp3 through transcriptional regulation of early growth response genes and E3 ubiquitin ligase genes, independently of thymic selection.