Companion Animals as Models for Inhibition of STAT3 and STAT5

The use of transgenic mouse models has revolutionized the study of many human diseases. However, murine models are limited in their representation of spontaneously arising tumors and often lack key clinical signs and pathological changes. Thus, a closer representation of complex human diseases is of high therapeutic relevance. Given the high failure rate of drugs at the clinical trial phase (i.e., around 90%), there is a critical need for additional clinically relevant animal models. Companion animals like cats and dogs display chronic inflammatory or neoplastic diseases that closely resemble the human counterpart. Cat and dog patients can also be treated with clinically approved inhibitors or, if ethics and drug safety studies allow, pilot studies can be conducted using, e.g., inhibitors of the evolutionary conserved JAK-STAT pathway. The incidence by which different types of cancers occur in companion animals as well as mechanisms of disease are unique between humans and companion animals, where one can learn from each other. Taking advantage of this situation, existing inhibitors of known oncogenic STAT3/5 or JAK kinase signaling pathways can be studied in the context of rare human diseases, benefitting both, the development of drugs for human use and their application in veterinary medicine

A Recurrent STAT5BN642H Driver Mutation in Feline Alimentary T Cell Lymphoma

Alimentary lymphomas arising from T cells are rare and aggressive malignancies in humans. In comparison, they represent the most common anatomical form of lymphoma in cats. Due to the low prevalence in humans, the underlying pathomechanism for these diseases is poorly characterised, limiting experimental analysis and therapeutic exploration. To date, activating mutations of the JAK/STAT core cancer pathway and particularly the STAT5B oncoprotein have been identified in human enteropathy-associated T cell lymphoma. Here, we describe a high homology of human and feline STAT3 and STAT5B proteins and strong conservation at the genomic level. Analysis of 42 samples of feline T cell alimentary lymphoma reveals broad activation of STAT3 and STAT5B. Screening for known activating mutations in STAT3 or STAT5B identifies the presence of the STAT5BN642H driver mutation in feline enteropathy-associated T cell lymphoma in 7 out of 42 (16.67%) samples in total. Regarding lymphoma subtypes, the majority of mutations with 5 out of 17 (29.41%) cases were found in feline enteropathy-associated lymphoma type II (EATL II). This identification of an oncogenic STAT5B driver mutation in felines recapitulates the genetic situation in the corresponding human disease, thereby establishing the cat as a potential new model for a rare and incurable human T cell disease

Ebf factors and MyoD cooperate to regulate muscle relaxation via Atp2a1

Jin, Saihong et al.Myogenic regulatory factors such as MyoD and Myf5 lie at the core of vertebrate muscle differentiation. However, E-boxes, the cognate binding sites for these transcription factors, are not restricted to the promoters/enhancers of muscle cell-specific genes. Thus, the specificity in myogenic transcription is poorly defined. Here we describe the transcription factor Ebf3 as a new determinant of muscle cell-specific transcription. In the absence of Ebf3 the lung does not unfold at birth, resulting in respiratory failure and perinatal death. This is due to a hypercontractile diaphragm with impaired Ca2+ efflux-related muscle functions. Expression of the Ca2+ pump Serca1 (Atp2a1) is downregulated in the absence of Ebf3, and its transgenic expression rescues this phenotype. Ebf3 binds directly to the promoter of Atp2a1 and synergises with MyoD in the induction of Atp2a1. In skeletal muscle, the homologous family member Ebf1 is strongly expressed and together with MyoD induces Atp2a1. Thus, Ebf3 is a new regulator of terminal muscle differentiation in the diaphragm, and Ebf factors cooperate with MyoD in the induction of muscle-specific genes. © 2014 Macmillan Publishers Limited.This work was supported by grants from the German Research Foundation (DFG, TRR54; FOR1586; FOR2033) and by a stipend of the Max Planck SocietyPeer Reviewe

EBF2 regulates osteoblast-dependent differentiation of osteoclasts.

Communication between bone-depositing osteoblasts and bone-resorbing osteoclasts is required for bone development and homeostasis. Here, we identify EBF2, a member of the early B cell factor (EBF) family of transcription factors that is expressed in osteoblast progenitors, as a regulator of osteoclast differentiation. We find that mice homozygous for a targeted inactivation of Ebf2 show reduced bone mass and an increase in the number of osteoclasts. These defects are accompanied by a marked downregulation of the osteoprotegerin (Opg) gene, encoding a RANK decoy receptor. EBF2 binds to sequences in the Opg promoter and transactivates the Opg promoter in synergy with the Wnt-responsive LEF1/TCF:β-catenin pathway. Taken together, these data identify EBF2 as a regulator of RANK-RANKL signaling and osteoblast-dependent differentiation of osteoclasts

No leakiness, high inducibility and strong expression of the transgene <i>in</i><i>vivo</i>.

<p>(A) Single cell suspensions from bone marrow of mice with the indicated genotypes were analysed for the expression of Gfp and the B cell marker B220. Gfp-positive cells are observed only in combination of the <i>vav</i>^<i>Cre</i> and the transgenic <i>Rosa26</i>^<i>RNAi</i> genotypes. All other genotypes show expression of B220, but a lack of Gfp. Note the slight shift in expression of Gfp from <i>Rosa26</i>^{<i>RNAi/+</i>} to <i>Rosa26</i>^{<i>RNAi/RNAi</i>}, due to the presence of two alleles of the transgene. Cell have been gated for FSC/SSC and as PI^-. (B) Statistical analysis of bone marrow cells stained and analysed as in A. Significant differences based on genotypes are observed only in combination of the <i>vav</i>^<i>Cre</i> and <i>Rosa26</i>^<i>RNAi</i> transgenes compared to all other genotypes; n=3-4; error bars=SD; ** p<0.01, *** p<0.001.</p

Expression of the transgene does not interfere with Ebf1 levels <i>in</i><i>vivo</i>.

<p>(A) Single cell suspensions from bone marrow of mice with the same genotypes as before were prepared and stained for B220/CD43 and CD24/BP1. Cells were gated in flow cytometry for FSC/SSC and as B220⁺CD43⁺ as indicated in the left panel in a representative example. The right panel shows the gates used for sorting of cells from fraction A (B220⁺CD43⁺CD24^-BP-1^-), B (B220⁺CD43⁺CD24⁺BP-1^-) and C (B220⁺CD43⁺CD24⁺BP-1⁺). These B cell fractions were sorted from all the genotypes used throughout this study. (B) Sorted cells from different B cell fractions of the indicated genotypes were subjected to qPCR analysis of Ebf1. All values were normalised to <i>HPRT</i> and the expression of Ebf1 in wild-type cells was set to 1 in each case; n=3-4; error bars=SD. (C) B220⁺CD43⁺ cells were sorted from total bone marrow of mice with the indicated genotypes and used for analysis of expression of Ebf1 by Western blot (Fractions A-C). Bone marrow from wild-type mice was depleted of B220⁺ cells (B220^- BMMNC) and used as negative control, β-actin is used as loading control. (D) Measurement of the amount of protein present in the B cell fractions and control as depicted in C using ImageJ; n=3-4; error bars=SD, ** p<0.01.</p

No relevant alterations in the adult cerebellum as a result of RNAi activation in the early cerebellar primordium.

<p>(A-B) Low magnification DAPI stainings of postnatal day 60 cerebella. Normal lobulation in the RNAi-expressing cerebellum (<i>Ebf2</i>^<i>Cre</i>::<i>Rosa</i>^{<i>26RNAi/+</i>}, panel B), compared to a Cre- control (A). (C-D) Sagittal sections of adult cerebella stained for the Purkinje cell (PC)-specific marker calbindin (CaBP, red) and for Gfp. RNAi-expressing PCs (Gfp⁺, panel D) develop normal somata and dendritic arbors positive for CaBP. Panel E (normal control) shows sagittally sectioned CaBP⁺ PCs from an <i>Ebf2</i>^<i>Cre</i>::<i>Rosa26</i>^{<i>YFP/YFP</i>} mouse. Size bars: B, 200 µm; E, 25 µm.</p