Pathogenesis of SHH medulloblastoma in mice

Medulloblastoma (MB) is the most common malignant brain tumor of childhood that comprises at least four molecularly distinct subgroups. One subgroup is characterized by aberrant Sonic hedgehog (SHH) signalling. Despite the ubiquitous activation of the SHH pathway within this subgroup, there is clear evidence that tumors with a SHH profile may vary in certain molecular and clinical aspects. Targeted therapies as a novel treatment modality for MB patients are especially intriguing for primary and relapsed SHH-MB. As more drugs targeting the Hh pathway become available and enter clinical trials, it is important to know how to stratify the patients for different drugs in order to maximize response rates and to prevent unnecessary treatment failures. In a first project, we aimed to characterize tumor localization in murine Shh-associated models. Using well established mouse models we show here that oncogenic transformation of cerebellar granule cell precursors at early developmental time points may result in the formation of midline and hemispheric medulloblastoma. On the other side, oncogenic transformation at later developmental time points exclusively results in the formation of hemispheric medulloblastoma. These data, which perfectly match to the recently published observations in human patients, indicate that granule neuron precursors are biologically distinct in different cerebellar compartments and that localization of SHH medulloblastoma is dependent on the time rather than the kind of genetic alteration. In a second project, we aimed to investigate the effect of the Wnt pathway on Shh-associated medulloblastoma. Previous studies were able to show that Wnt/ß-Catenin activation might be able to inhibit Shh-associated medulloblastoma growth through downregulation of the Shh pathway. Considering a possible therapeutic approach, in vitro treatments with lithium chloride, a well-known Wnt/ß-Catenin agonist, were carried out. Lithium chloride treatment in vitro resulted in a decrease of granule neuron precursor and tumor cell viability. In the third project, we aimed to better understand the mechanisms in MB of primary and secondary resistance to drugs targeting the Hh pathway. To this end, we generated new mouse models, characterized and used these and already existing genetically engineered mouse models (GEMMs). We aimed to further expand our pre-existing repertoire of murine SHH-activated MB models in order to have a spectrum of tumors that are driven by mutations at different levels of the Hh pathway, such as PTCH1, SMO, or MYCN, and with different combinations of other mutations in additional pathways that may co-operate with HH signalling (e.g. TP53 or PIK3CA). The mouse strains Math1-creERT2::lsl-SmoM2Fl/+, Math1-creERT2::lsl-SmoM2Fl/+ lsl-Pik3caFl/+, Math1-creERT2::Ptch1Fl/Fl, Math1-creERT2::Ptch1Fl/Fl lsl-Pik3caFl/+, and Math1-creERT2::Ptch1Fl/Fl lsl-Pik3caFl/Fl developed Shh-associated medulloblastoma. Our mouse model with a MYCN mutation did not develop any tumor, neither as such nor with an additional TP53 mutation. Math1-creERT2::lsl-SmoM2Fl/+, Math1-creERT2::lsl-SmoM2Fl/+ lsl-Pik3caFl/+, and Math1-creERT2::Ptch1Fl/Fl mice were then treated with the SMO inhibitor LDE225, that is already used in clinical trials. Math1-creERT2::lsl-SmoM2Fl/+ and Math1-creERT2::Ptch1Fl/Fl mice first benefit from the treatment, but then also die due to symptoms of the tumor, whereas Math1-creERT2::lsl-SmoM2Fl/+ lsl-Pik3caFl/+ mice did not show a better prognosis for survival at all. None the less, proliferation was reduced in all tumors treated with LDE225

Canonical Wnt Signaling Drives Tumor-Like Lesions from Sox2-Positive Precursors of the Murine Olfactory Epithelium.

Canonical Wnt signaling is known to promote proliferation of olfactory stem cells. In order to investigate the effects of a constitutive activation of Wnt signaling in Sox2-positive precursor cells of the olfactory epithelium, we used transgenic mice that allowed an inducible deletion of exon 3 of the Ctnnb1 gene, which is responsible for the phosphorylation and degradation of Ctnnb1 protein. After induction of aberrant Wnt activation by Ctnnb1 deletion at embryonic day 14, such mice developed tumor-like lesions in upper parts of the nasal cavity. We still observed areas of epithelial hyperplasia within the olfactory epithelium following early postnatal Wnt activation, but the olfactory epithelial architecture remained unaffected in most parts when Wnt was activated at postnatal day 21 or later. In summary, our results suggest an age-dependent tumorigenic potential of aberrant Wnt signaling in the olfactory epithelium of mice

Additional immunohistochemical staining of tumor-like lesions in embryonically induced mutant mice suggest similarity to human olfactory neuroblastoma (hONB), but distinctness from human sinonasal haemangiopericytoma (sHPC).

<p>The expression of markers of neuroendocrine differentiation as CD56 and Chromogranin A and positivity for S100 are crucial requirements for the proper diagnosis of hONB and can be detected in OE mouse tumor-like lesions (B, D, F), but are found to be absent in the native mouse OE (A, C, E). SMA is known to be widely diffuse positive in sinonasal haemangiopericytoma but does not homogenously stain cells in our mouse tumor-like lesions (H) or native mouse OE (G). Scale bars equate to 25 μm.</p

The <i>Sox2-CreER</i>^<i>T2</i> mouse is a suitable driver strain for studying gene function in the mouse olfactory epithelium (OE).

<p>At embryonic day 14.5 of mouse development (E14.5) the transcription factor Sox2 is already expressed in basal and apical progenitor cells of the olfactory epithelium (A). The mouse breeding scheme used in this study is displayed in panel B. Model induction via single tamoxifen application at E14.5 targets the whole progeny of the OE structure present at the day of birth (P0) as demonstrated in a <i>Sox2-creER</i>^<i>T2</i>::<i>tdTomato</i> reporter strain (D). These targeted cells comprise Sox2-positive stem cells at the basal site of the OE (arrows, C-E), non-neuronal apical Sox2-positive sustentacular cells (nuclei of sustentacular cells delineated by dashed line, C-E) as well as Tuj-1-positive neuronal cells of the OE (a Tuj1/RFP-double-positive cell is depicted in high power insets, F-H). Single embryonic tamoxifen injection of <i>Sox2-creER</i>^<i>T2</i>::<i>tdTomato</i> animals is sufficient to cover the whole mature OE in adult mice as shown for P28 (J; specific RFP detection compared to control in I). Scale bars equate to 500 μm in I-J, 25 μm in A and 10 μm in C-H.</p

Early postnatally activated aberrant Wnt signaling in <i>Sox2-creER</i>^<i>T2</i>::<i>Ctnnb1(ex3)</i>^<i>Fl/+</i> mice leads to the formation of epithelial hyperplasia within the OE.

<p>Tamoxifen application in <i>Sox2-creER</i>^<i>T2</i>::<i>Ctnnb1(ex3)</i>^<i>Fl/+</i> mice at postnatal day 7 (P7) or 14 (P14) leads to the formation of OE hyperplasia (A, E, OE of control mice; B, F, higher magnification of framed areas in A, E showing normal OE; C, hyperplasia in P7-induced mutant mice; D, higher magnification of framed area in C; G, multiple areas of hyperplasia in P14-induced mutant mice captured on different slice levels; H, higher magnification of framed area in G). IHC staining patterns for Ki67, Sox2, ß-Catenin and Mash1 of exemplary areas of hyperplasia in P14-induced mutant mice are comparable to control OE, indicating a grossly normal cell differentiation and preserved OE polarity in such areas (I, J, M, N, IHC stains of control mice OE; K, L, O, P, respective IHC stains of hyperplasia; insets display high power magnifications of framed areas in I-P and exemplarily highlight positive cells for the respective marker). At postnatal day 21 (P21) tamoxifen-induced mutant mice do not develop those alterations anymore (Q-T). Scale bars equate to 500 μm in A, C, E, G, Q and S, equate to 100 μm in B, D, F, H, R and T, equate to 50 μm in I-P, and equate to 10 μm in insets.</p

Embryonically induced mutant mice present with a failure to thrive and die prematurely when compared to controls.

<p>At E14.5 tamoxifen-induced <i>Sox2-creER</i>^<i>T2</i>::<i>Ctnnb1(ex3)</i>^<i>Fl/+</i> mice develop a marked failure to thrive compared to controls (B vs. A) and show significant deficits in body size (C) and body weight (D) at their time of death. Mutant mice had to be sacrificed prematurely between postnatal days 21–28 (E).</p

TCF4 (E2-2) harbors tumor suppressive functions in SHH medulloblastoma

The TCF4 gene encodes for the basic helix–loop–helix transcription factor 4 (TCF4), which plays an important role in the development of the central nervous system (CNS). Haploinsufficiency of TCF4 was found to cause Pitt-Hopkins syndrome (PTHS), a severe neurodevelopmental disorder. Recently, the screening of a large cohort of medulloblastoma (MB), a highly aggressive embryonal brain tumor, revealed almost 20% of adult patients with MB of the Sonic hedgehog (SHH) subtype carrying somatic TCF4 mutations. Interestingly, many of these mutations have previously been detected as germline mutations in patients with PTHS. We show here that overexpression of wild-type TCF4 in vitro significantly suppresses cell proliferation in MB cells, whereas mutant TCF4 proteins do not to the same extent. Furthermore, RNA sequencing revealed significant upregulation of multiple well-known tumor suppressors upon expression of wild-type TCF4. In vivo, a prenatal knockout of Tcf4 in mice caused a significant increase in apoptosis accompanied by a decreased proliferation and failed migration of cerebellar granule neuron precursor cells (CGNP), which are thought to be the cells of origin for SHH MB. In contrast, postnatal in vitro and in vivo knockouts of Tcf4 with and without an additional constitutive activation of the SHH pathway led to significantly increased proliferation of CGNP or MB cells. Finally, publicly available data from human MB show that relatively low expression levels of TCF4 significantly correlate with a worse clinical outcome. These results not only point to time-specific roles of Tcf4 during cerebellar development but also suggest a functional linkage between TCF4 mutations and the formation of SHH MB, proposing that TCF4 acts as a tumor suppressor during postnatal stages of cerebellar development

Identification of infants with increased type 1 diabetes genetic risk for enrollment into Primary Prevention Trials—GPPAD-02 study design and first results

Primary prevention of type 1 diabetes (T1D) requires intervention in genetically at-risk infants. The Global Platform for the Prevention of Autoimmune Diabetes (GPPAD) has established a screening program, GPPAD-02, that identifies infants with a genetic high risk of T1D, enrolls these into primary prevention trials, and follows the children for beta-cell autoantibodies and diabetes. Genetic testing is offered either at delivery, together with the regular newborn testing, or at a newborn health care visits before the age of 5 months in regions of Germany (Bavaria, Saxony, Lower Saxony), UK (Oxford), Poland (Warsaw), Belgium (Leuven), and Sweden (Region Skåne). Seven clinical centers will screen around 330 000 infants. Using a genetic score based on 46 T1D susceptibility single-nucleotide polymorphisms (SNPs) or three SNPS and a first-degree family history for T1D, infants with a high (>10%) genetic risk for developing multiple beta-cell autoantibodies by the age of 6 years are identified. Screening from October 2017 to December 2018 was performed in 50 669 infants. The prevalence of high genetic risk for T1D in these infants was 1.1%. Infants with high genetic risk for T1D are followed up and offered to participate in a randomized controlled trial aiming to prevent beta-cell autoimmunity and T1D by tolerance induction with oral insulin. The GPPAD-02 study provides a unique path to primary prevention of beta-cell autoimmunity in the general population. The eventual benefit to the community, if successful, will be a reduction in the number of children developing beta-cell autoimmunity and T1D

Characterization of pancreatic glucagon-producing tumors and pituitary gland tumors in transgenic mice overexpressing MYCN in hGFAP-positive cells

Amplification or overexpression of MYCN is involved in development and maintenance of multiple malignancies. A subset of these tumors originates from neural precursors, including the most aggressive forms of the childhood tumors, neuroblastoma and medulloblastoma. In order to model the spectrum of MYCNdriven neoplasms in mice, we transgenically overexpressed MYCN under the control of the human GFAP-promoter that, among other targets, drives expression in neural progenitor cells. However, LSL-MYCN;hGFAP-Cre double transgenic mice did neither develop neural crest tumors nor tumors of the central nervous system, but presented with neuroendocrine tumors of the pancreas and, less frequently, the pituitary gland. Pituitary tumors expressed chromogranin A and closely resembled human pituitary adenomas. Pancreatic tumors strongly produced and secreted glucagon, suggesting that they derived from glucagon- and GFAP-positive islet cells. Interestingly, 3 out of 9 human pancreatic neuroendocrine tumors expressed MYCN, supporting the similarity of the mouse tumors to the human system. Serial transplantations of mouse tumor cells into immunocompromised mice confirmed their fully transformed phenotype. MYCN-directed treatment by AuroraA- or Brd4-inhibitors resulted in significantly decreased cell proliferation in vitro and reduced tumor growth in vivo. In summary, we provide a novel mouse model for neuroendocrine tumors of the pancreas and pituitary gland that is dependent on MYCN expression and that may help to evaluate MYCN-directed therapies