Traces of Canine Inflammatory Bowel Disease Reflected by Intestinal Organoids.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition that affects humans and several domestic animal species, including cats and dogs. In this study, we have analyzed duodenal organoids derived from canine IBD patients using quantitative proteomics. Our objective was to investigate whether these organoids show phenotypic traits of the disease compared with control organoids obtained from healthy donors. To this aim, IBD and control organoids were subjected to quantitative proteomics analysis via liquid chromatography-mass spectrometry. The obtained data revealed notable differences between the two groups. The IBD organoids exhibited several alterations at the levels of multiple proteins that are consistent with some known IBD alterations. The observed phenotype in the IBD organoids to some degree mirrors the corresponding intestinal condition, rendering them a compelling approach for investigating the disease and advancing drug exploration. Additionally, our study revealed similarities to some human IBD biomarkers, further emphasizing the translational and comparative value of dogs for future investigations related to the causes and treatment of IBD. Relevant proteins such as CALU, FLNA, MSN and HMGA2, which are related to intestinal diseases, were all upregulated in the IBD duodenal organoids. At the same time, other proteins such as intestinal keratins and the mucosal immunity PIGR were depleted in these IBD organoids. Based on these findings, we propose that these organoids could serve as a valuable tool for evaluating the efficacy of therapeutic interventions against canine IBD.

Neutralising Effects of Different Antibodies on Clostridioides difficile Toxins TcdA and TcdB in a Translational Approach.

Given the high prevalence of intestinal disease in humans and animals, there is a strong need for clinically relevant models recapitulating gastrointestinal systems, ideally replacing in vivo models in accordance with the principles of the 3R. We established a canine organoid system and analysed the neutralising effects of recombinant versus natural antibodies on Clostridioides difficile toxins A and B in this in vitro system. Sulforhodamine B cytotoxicity assays in 2D and FITC-dextran barrier integrity assays on basal-out and apical-out organoids revealed that recombinant, but not natural antibodies, effectively neutralised C. difficile toxins. Our findings emphasise that canine intestinal organoids can be used to test different components and suggest that they can be further refined to also mirror complex interactions between the intestinal epithelium and other cells.

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.

Cell Metabolism Control Through O-GlcNAcylation of STAT5: A Full or Empty Fuel Tank Makes a Big Difference for Cancer Cell Growth and Survival.

O-GlcNAcylation is a post-translational modification that influences tyrosine phosphorylation in healthy and malignant cells. O-GlcNAc is a product of the hexosamine biosynthetic pathway, a side pathway of glucose metabolism. It is essential for cell survival and proper gene regulation, mirroring the metabolic status of a cell. STAT3 and STAT5 proteins are essential transcription factors that can act in a mutational context-dependent manner as oncogenes or tumor suppressors. They regulate gene expression for vital processes such as cell differentiation, survival, or growth, and are also critically involved in metabolic control. The role of STAT3/5 proteins in metabolic processes is partly independent of their transcriptional regulatory role, but is still poorly understood. Interestingly, STAT3 and STAT5 are modified by O-GlcNAc in response to the metabolic status of the cell. Here, we discuss and summarize evidence of O-GlcNAcylation-regulating STAT function, focusing in particular on hyperactive STAT5A transplant studies in the hematopoietic system. We emphasize that a single O-GlcNAc modification is essential to promote development of neoplastic cell growth through enhancing STAT5A tyrosine phosphorylation. Inhibition of O-GlcNAcylation of STAT5A on threonine 92 lowers tyrosine phosphorylation of oncogenic STAT5A and ablates malignant transformation. We conclude on strategies for new therapeutic options to block O-GlcNAcylation in combination with tyrosine kinase inhibitors to target neoplastic cancer cell growth and survival.

Correction: The ratio of STAT1 to STAT3 expression is a determinant of colorectal cancer growth.

[This corrects the article DOI: 10.18632/oncotarget.9315.].

Pharmacologic inhibition of STAT5 in acute myeloid leukemia.

The transcription factor STAT5 is an essential downstream mediator of many tyrosine kinases (TKs), particularly in hematopoietic cancers. STAT5 is activated by FLT3-ITD, which is a constitutively active TK driving the pathogenesis of acute myeloid leukemia (AML). Since STAT5 is a critical mediator of diverse malignant properties of AML cells, direct targeting of STAT5 is of significant clinical value. Here, we describe the development and preclinical evaluation of a novel, potent STAT5 SH2 domain inhibitor, AC-4-130, which can efficiently block pathological levels of STAT5 activity in AML. AC-4-130 directly binds to STAT5 and disrupts STAT5 activation, dimerization, nuclear translocation, and STAT5-dependent gene transcription. Notably, AC-4-130 substantially impaired the proliferation and clonogenic growth of human AML cell lines and primary FLT3-ITD+ AML patient cells in vitro and in vivo. Furthermore, AC-4-130 synergistically increased the cytotoxicity of the JAK1/2 inhibitor Ruxolitinib and the p300/pCAF inhibitor Garcinol. Overall, the synergistic effects of AC-4-130 with TK inhibitors (TKIs) as well as emerging treatment strategies provide new therapeutic opportunities for leukemia and potentially other cancers.

Cannabinoid Receptors Are Overexpressed in CLL but of Limited Potential for Therapeutic Exploitation.

The cannabinoid receptors 1 and 2 (CNR1&2) are overexpressed in a variety of malignant diseases and cannabinoids can have noteworthy impact on tumor cell viability and tumor growth. Patients diagnosed with chronic lymphocytic leukemia (CLL) present with very heterogeneous disease characteristics translating into highly differential risk properties. To meet the urgent need for refinement in risk stratification at diagnosis and the search for novel therapies we studied CNR expression and response to cannabinoid treatment in CLL. Expression levels of CNR1&2 were determined in 107 CLL patients by real-time PCR and analyzed with regard to prognostic markers and survival. Cell viability of primary CLL cells was determined in suspension and co-culture after incubation in increasing cannabinoid concentrations under normal and reduced serum conditions and in combination with fludarabine. Impact of cannabinoids on migration of CLL cells towards CXCL12 was determined in transwell plates. We found CNR1&2 to be overexpressed in CLL compared to healthy B-cells. Discriminating between high and low expressing subgroups, only high CNR1 expression was associated with two established high risk markers and conferred significantly shorter overall and treatment free survival. Viability of CLL primary cells was reduced in a dose dependent fashion upon incubation with cannabinoids, however, healthy cells were similarly affected. Under serum reduced conditions, no significant differences were observed within suspension and co-culture, respectively, however, the feeder layer contributed significantly to the survival of CLL cells compared to suspension culture conditions. No significant differences were observed when treating CLL cells with cannabinoids in combination with fludarabine. Interestingly, biologic activity of cannabinoids was independent of both CNR1&2 expression. Finally, we did not observe an inhibition of CXCL12-induced migration by cannabinoids. In contrast to other tumor entities, our data suggest a limited usability of cannabinoids for CLL therapy. Nonetheless, we could define CNR1 mRNA expression as novel prognostic marker.

The ratio of STAT1 to STAT3 expression is a determinant of colorectal cancer growth.

The role of STAT1 and STAT3 for colorectal carcinoma (CRC) development and progression is controversial. We evaluated 414 CRC patient samples on tissue microarrays for differential expression of STAT1 and STAT3 protein levels and correlated ratios with clinical parameters. Concomitant absence of nuclear STAT1 and STAT3 expression was associated with significantly reduced median survival by ≥33 months (p=0.003). To gain insight into underlying mechanisms, we generated four CRC cell lines with STAT3 knockdown. The cell lines harbor different known mutational drivers and were xenografted into SCID mice to analyze the influence of STAT3 on their tumor growth behavior. Experimental downregulation of STAT3 expression had differential, cell-line specific effects on STAT1 expression levels. STAT1 consistently showed nuclear localization irrespective of its tyrosine phosphorylation status. Two characteristic STAT1/3 expression patterns with opposite growth behavior could be distinguished: cell lines with a low STAT1/high STAT3 ratio showed faster tumor growth in xenografts. In contrast, xenografts of cell lines showing high STAT1 and low STAT3 levels grew slower. Importantly, these ratios reflected clinical outcome in CRC patients as well. We conclude that the ratio of STAT1 to STAT3 expression is a key determinant of CRC progression and that STAT1 counteracts pro-tumorigenic STAT3 signaling. Thus, we suggest that the STAT3/STAT1 ratios are better clinical predictors in CRC as compared to STAT3 or STAT1 levels alone.

SAR-guided development and characterization of a potent antitumor compound toward B-cell neoplasms with no detectable cytotoxicity toward healthy cells.

Acute hematological diseases (leukemias and aggressive lymphomas) can be cured in approximately half of the patients, while the other patients die from their disease. Chronic leukemias and indolent lymphomas can be well controlled for years in most cases. However, the cure rate of these patients is low and the course of the disease is characterized by frequent recurrence. Therefore, novel agents for monotherapies or combination therapies still need to be explored. The presented study describes the identification of the chalcone derivative 15 on different types of human malignant cells of the lymphoid and myeloid lineage. Further experiments performed with compound 15 on peripheral blood mononuclear cells (PBMCs) of chronic lymphocytic leukemia (CLL) patients clearly stated a higher cytotoxicity in PBMC from CLL patients compared to healthy donors (HD). The newly identified chalcone derivative 15 showed a higher therapeutic potential than fludarabine, a drug already in use in lymphoma treatment.