How to Obtain a Mega-Intestine with Normal Morphology: In Silico Modelling of Postnatal Intestinal Growth in a Cd97-Transgenic Mouse.

Intestinal cylindrical growth peaks in mice a few weeks after birth, simultaneously with crypt fission activity. It nearly stops after weaning and cannot be reactivated later. Transgenic mice expressing Cd97/Adgre5 in the intestinal epithelium develop a mega-intestine with normal microscopic morphology in adult mice. Here, we demonstrate premature intestinal differentiation in Cd97/Adgre5 transgenic mice at both the cellular and molecular levels until postnatal day 14. Subsequently, the growth of the intestinal epithelium becomes activated and its maturation suppressed. These changes are paralleled by postnatal regulation of growth factors and by an increased expression of secretory cell markers, suggesting growth activation of non-epithelial tissue layers as the origin of enforced tissue growth. To understand postnatal intestinal growth mechanistically, we study epithelial fate decisions during this period with the use of a 3D individual cell-based computer model. In the model, the expansion of the intestinal stem cell (SC) population, a prerequisite for crypt fission, is largely independent of the tissue growth rate and is therefore not spontaneously adaptive. Accordingly, the model suggests that, besides the growth activation of non-epithelial tissue layers, the formation of a mega-intestine requires a released growth control in the epithelium, enabling accelerated SC expansion. The similar intestinal morphology in Cd97/Adgre5 transgenic and wild type mice indicates a synchronization of tissue growth and SC expansion, likely by a crypt density-controlled contact inhibition of growth of intestinal SC proliferation. The formation of a mega-intestine with normal microscopic morphology turns out to originate in changes of autonomous and conditional specification of the intestinal cell fate induced by the activation of Cd97/Adgre5.

Fighting Against Promoter DNA Hyper-Methylation: Protective Histone Modification Profiles of Stress-Resistant Intestinal Stem Cells.

Aberrant DNA methylation in stem cells is a hallmark of aging and tumor development. Recently, we have suggested that promoter DNA hyper-methylation originates in DNA repair and that even successful DNA repair might confer this kind of epigenetic long-term change. Here, we ask for interrelations between promoter DNA methylation and histone modification changes observed in the intestine weeks after irradiation and/or following Msh2 loss. We focus on H3K4me3 recruitment to the promoter of H3K27me3 target genes. By RNA- and histone ChIP-sequencing, we demonstrate that this recruitment occurs without changes of the average gene transcription and does not involve H3K9me3. Applying a mathematical model of epigenetic regulation of transcription, we show that the recruitment can be explained by stronger DNA binding of H3K4me3 and H3K27me3 histone methyl-transferases as a consequence of lower DNA methylation. This scenario implicates stable transcription despite of H3K4me3 recruitment, in agreement with our RNA-seq data. Following several kinds of stress, including moderate irradiation, stress-sensitive intestinal stem cell (ISCs) are known to become replaced by more resistant populations. Our simulation results suggest that the stress-resistant ISCs are largely protected against promoter hyper-methylation of H3K27me3 target genes.

Linking stem cell function and growth pattern of intestinal organoids.

Intestinal stem cells (ISCs) require well-defined signals from their environment in order to carry out their specific functions. Most of these signals are provided by neighboring cells that form a stem cell niche, whose shape and cellular composition self-organize. Major features of this self-organization can be studied in ISC-derived organoid culture. In this system, manipulation of essential pathways of stem cell maintenance and differentiation results in well-described growth phenotypes. We here provide an individual cell-based model of intestinal organoids that enables a mechanistic explanation of the observed growth phenotypes. In simulation studies of the 3D structure of expanding organoids, we investigate interdependences between Wnt- and Notch-signaling which control the shape of the stem cell niche and, thus, the growth pattern of the organoids. Similar to in vitro experiments, changes of pathway activities alter the cellular composition of the organoids and, thereby, affect their shape. Exogenous Wnt enforces transitions from branched into a cyst-like growth pattern; known to occur spontaneously during long term organoid expansion. Based on our simulation results, we predict that the cyst-like pattern is associated with biomechanical changes of the cells which assign them a growth advantage. The results suggest ongoing stem cell adaptation to in vitro conditions during long term expansion by stabilizing Wnt-activity. Our study exemplifies the potential of individual cell-based modeling in unraveling links between molecular stem cell regulation and 3D growth of tissues. This kind of modeling combines experimental results in the fields of stem cell biology and cell biomechanics constituting a prerequisite for a better understanding of tissue regeneration as well as developmental processes.

Angiogenic inflammation and formation of necrosis in the tumor microenvironment influence patient survival after radical surgery for de novo hepatocellular carcinoma in non-cirrhosis.

BACKGROUND: Tumor escape mechanisms mediated in the tumor microenvironment can significantly reduce the capacity of the anti-tumor function of the immune system. TIE2-expressing monocytes (TEMs), related angiopoietins, and tumor necrosis are considered to have a key role in this process. We aimed to investigate the abundance and clinical significance of these biomarkers in hepatocellular carcinoma (HCC). METHODS: In this retrospective study, 58 HCC patients received surgery with a curative intent. The abundance of TEMs, angiopoietin-1 and -2 were detected in tumor specimens of the HCC patients (n = 58), and together with the occurrence of histologic tumor necrosis, were associated with established clinicopathological characteristics and survival. RESULTS: Patients with HCC characterized by necrosis and TEMs revealed reduced both overall survival and recurrence-free survival (all p < 0.05). Angiopoietins and TEMs were associated with metastatic and recurrent HCC. Furthermore, the formation of histologic tumor necrosis was associated with advanced tumor stage and density of TEMs (all p < 0.05). CONCLUSIONS: Histologic tumor necrosis, TEMs, and related angiopoietins were associated with multiple HCC parameters and patient survival. The tumor necrosis-TEM-angiopoietin axis may offer a novel diagnostic modality to predict patient outcome after surgery for HCC.

Immunologic cellular characteristics of the tumour microenvironment of hepatocellular carcinoma drive patient outcomes.

BACKGROUND: Anti-tumour immune competence has an impact in hepatocarcinogenesis and success of anti-cancer therapies. Tumour-infiltrating lymphocytes (TILs) and monocytes/macrophages (TAMs) are proposed to have significance in cancer. However, there is only limited data concerning their impact on patient outcome and survival in hepatocellular carcinoma (HCC). METHODS: Frequencies of CD68+, CD163+ M2-polarized TAMs and TILs were measured in de novo HCC tumours in non-cirrhosis (n = 58) using immunohistology and correlated to patients' clinicopathological characteristics and survival rates. RESULTS: Patients with tumours marked by appearance of TILs and CD68+ TAMs showed an improved 1-, 3- and 5-year recurrence-free survival (all p ≤ 0.05). CD68+ TAMs were associated with reduced incidence of recurrent and multifocal disease. Conversely, CD163+ TAMs were associated with multifocal HCC and lymphangiosis carcinomatosa (all p ≤ 0.05). CONCLUSIONS: TILs and CD68+ TAMs are associated with multiple tumour characteristics and patient survival in HCC. However, there is only scarce data about the biology underlying their mechanistic involvement in human tumour progression. Thus, experimental data on functional links might help develop novel immunologic checkpoint inhibitor targets for liver cancer.

Increased circulating chemerin in patients with advanced carotid stenosis.

BACKGROUND: Chemerin is an adipokine which plays a crucial role in atherosclerosis. Here, we examined whether circulating chemerin is enhanced in patients with advanced carotid stenosis. METHODS: Chemerin was quantified in 178 patients prior to carotid end arterectomy (CEA) and in age- and gender-matched controls (n = 163). Chemerin levels were related to anthropometric, clinical and metabolic characteristics of the patients. RESULTS: Chemerin levels were higher in patients compared to controls (p <  0.001). Chemerin correlated to parameters associated with inflammation such as C-reactive protein (CRP, p <  0.001), leukocyte blood count (p <  0.001) and circulating TNF-α (p = 0.004) in the patients. Chemerin levels did not differ between asymptomatic (n = 93) and symptomatic patients who experienced an ischemic event within 6 months prior to CEA (n = 85). However, in the case of high-grade carotid stenosis (≥ 90%), chemerin levels were higher in symptomatic (n = 44) compared to asymptomatic patients (n = 41, p = 0.014). Chemerin was increased in patients with (n = 50) compared to patients without (n = 128) coronary artery disease (CAD, p = 0.002). A high level of chemerin increases the risk for CAD in patients (p = 0.0013). CONCLUSIONS: Circulating chemerin is increased and correlates to inflammatory parameters in patients with advanced carotid stenosis.

International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors.

The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.

Adhesion GPCRs in Tumorigenesis.

Alterations in the homeostasis of several adhesion GPCRs (aGPCRs) have been observed in cancer. The main cellular functions regulated by aGPCRs are cell adhesion, migration, polarity, and guidance, which are all highly relevant to tumor cell biology. Expression of aGPCRs can be induced, increased, decreased, or silenced in the tumor or in stromal cells of the tumor microenvironment, including fibroblasts and endothelial and/or immune cells. For example, ADGRE5 (CD97) and ADGRG1 (GPR56) show increased expression in many cancers, and initial functional studies suggest that both are relevant for tumor cell migration and invasion. aGPCRs can also impact the regulation of angiogenesis by releasing soluble fragments following the cleavage of their extracellular domain (ECD) at the conserved GPCR-proteolytic site (GPS) or other more distal cleavage sites as typical for the ADGRB (BAI) family. Interrogation of in silico cancer databases suggests alterations in other aGPCR members and provides the impetus for further exploration of their potential role in cancer. Integration of knowledge on the expression, regulation, and function of aGPCRs in tumorigenesis is currently spurring the first preclinical studies to examine the potential of aGPCR or the related pathways as therapeutic targets.

Perioperative risk factors in patients with a femoral neck fracture - influence of 25-hydroxyvitamin D and C-reactive protein on postoperative medical complications and 1-year mortality.

BACKGROUND: This study examined the association of 25-hydroxyvitamin D (25(OH)D) and C-reactive protein (CRP) with postoperative medical complications and one year mortality of elderly patients sustaining a low-energy cervical hip fracture scheduled for surgery. We hypothesized that vitamin D deficiency and CRP in these patients might be associated with an increased 1-year mortality. METHODS: The prospective single-center cohort study included 209 patients with a low-energy medial femoral neck fracture; 164 women aged over 50 years and 45 men aged over 60 years. Referring to 1-year mortality and postoperative medical complications multiple logistic regression analysis including 10 co-variables (age, sex, BMI, ASA, creatinine, CRP, leukocytes hemoglobin, 25(OH)D, vitamin D supplementation at follow-up) was performed. RESULTS: Vitamin D deficiency was prevalent in 87 % of all patients. In patients with severe (<10 ng/ml) and moderate (10-20 ng/ml) vitamin D deficiency one year mortality was 29 % and 13 %, respectively, compared to 9 % in patients with > 20 ng/ml 25(OH)D levels (p =0.027). Patients with a mild (CRP 10-39.9 mg/l) or active inflammatory response (CRP ≥ 40 mg/l) showed a higher one year mortality of 33 % and 40 % compared to 16 % in patients with no (CRP < 10 mg/l) inflammatory response (p = 0.002). Multiple logistic regression analysis identified CRP (OR 1.01, 95 % CI 1.00-1.02; p = 0.007), but not 25(OH)D (OR 0.97, 95 % CI 0.89-1.05; p = 0.425) as an independent predictor for one year mortality. 20 % of patients suffered in-hospital postoperative medical complications (i.e. pneumonia, thromboembolic events, etc.). 25(OH)D (OR 0.89, 95 % CI 0.81-0.97; p = 0.010), but not CRP (OR 1.01, 95 % CI 1.00-1.02; p = 0.139), was identified as an independent risk factor. CONCLUSION: In elderly patients with low-energy cervical hip fracture, 25(OH)D is independently associated with postoperative medical complications and CRP is an independent predictor of one year mortality.

Preclinical good laboratory practice-compliant safety study to evaluate biodistribution and tumorigenicity of a cartilage advanced therapy medicinal product (ATMP).

BACKGROUND: The clinical development of advanced therapy medicinal products (ATMPs), a new class of drugs, requires initial safety studies that deviate from standard non-clinical safety protocols. The study provides a strategy to address the safety aspects of biodistribution and tumorigenicity of ATMPs under good laboratory practice (GLP) conditions avoiding cell product manipulation. Moreover, the strategy was applied on a human ATMP for cartilage repair. METHODS: The testing strategy addresses biodistribution and tumorigenicity using a multi-step analysis without any cell manipulation to exclude changes of test item characteristics. As a safeguard measurement for meeting regulatory expectations, the project design and goals were discussed continuously with the regulatory authority using a staggered scientific advice concept. Subsequently, the strategy was applied to co.don chondrosphere® (huChon spheroid), a tissue-engineered matrix-free ATMP of human normal chondrocytes. In both the biodistribution and tumorigenicity studies, huChon spheroids were implanted subcutaneously into 40 immunodeficient mice. Biodistribution was studied 1 month after implantation. A skin disc containing the huChon spheroid, two surrounding skin rings and selected organs were analyzed by validated, gender-specific, highly-sensitive triplex qPCR and by immunohistochemistry (IHC). RESULTS: No human DNA was detected in distant skin rings and analyzed organs. IHC revealed no direct or indirect indications of cell migration. Tumorigenicity was assessed 6 months after huChon spheroid implantation by palpation, macroscopic inspection, histology and IHC. No mice from the huChon spheroid group developed a tumor at the implantation site. In two mice, benign tumors were detected that were negative for HLA-ABC, suggesting that they were of spontaneous murine origin. CONCLUSIONS: In summary, the presented strategy using a multi-step analysis was confirmed to be suitable for safety studies of ATMPs.

Thy-1 (CD90) is an interacting partner for CD97 on activated endothelial cells.

Leukocyte recruitment in response to inflammatory signals is governed, in part, by binding to Thy-1 (CD90) on activated endothelial cells (EC). In this study, we characterized the adhesion G-protein coupled receptor CD97, present on peripheral myeloid cells, as a novel interacting partner for Thy-1. CD97 was upregulated on polymorphonuclear cells (PMNC) of patients with psoriasis. In psoriatic skin lesions, CD97(+) myeloid cells colocalized with Thy-1(+) EC of small vessels in microabscesses, suggesting an interaction between CD97 and Thy-1 that was further examined by adhesion and protein-binding assays. PMNC and cell lines stably overexpressing CD97 adhered specifically to Thy-1(+)-activated human dermal EC, Thy-1(+) CHO cells, and immobilized Thy-1 protein. Binding of the CD97(+) CHO clones correlated with their CD97 expression level. Soluble CD97 bound specifically to immobilized Thy-1 protein, as well as Thy-1(+)-activated EC and CHO cells. In all assays, cellular adhesion or protein binding was blocked partially by CD97 and Thy-1-blocking mAb. Our data suggested that CD97 interacts via its stalk with Thy-1 because mAb directed to the stalk of CD97 showed stronger blocking compared with mAb to its epidermal growth factor-like domains, and binding was calcium independent. Moreover, soluble CD97 without the stalk and soluble EMR2, containing highly homologous epidermal growth factor-like domains but a different stalk, failed to bind. In summary, binding of leukocytes to activated endothelium mediated by the interaction of CD97 with Thy-1 is involved in firm adhesion of PMNC during inflammation and may play a role in the regulation of leukocyte trafficking to inflammatory sites.

Correction: Winkler et al. The Adhesion G-Protein-Coupled Receptor GPR115/ADGRF4 Regulates Epidermal Differentiation and Associates with Cytoskeletal KRT1. Cells 2022, 11, 3151.

In the original publication [...].

The Posttraumatic Increase in the Adhesion of GPCR EMR2/ADGRE2 to Circulating Neutrophils Is Not Related to Injury Severity.

Trauma triggers a rapid innate immune response to aid the clearance of damaged/necrotic cells and their released damage-associated molecular pattern (DAMP). Here, we monitored the expression of EMR2/ADGRE2, involved in the functional regulation of innate immune cells, on circulating neutrophils in very severely and moderately/severely injured patients up to 240 h after trauma. Notably, neutrophilic EMR2 showed a uniform, injury severity- and type of injury-independent posttraumatic course in all patients. The percentage of EMR2+ neutrophils and their EMR2 level increased and peaked 48 h after trauma. Afterwards, they declined and normalized in some, but not all, patients. Circulating EMR2+ compared to EMR2- neutrophils express less CD62L and more CD11c, a sign of activation. Neutrophilic EMR2 regulation was verified in vitro. Remarkably, it increased, depending on extracellular calcium, in controls as well. Cytokines, enhanced in patients immediately after trauma, and sera of patients did not further affect this neutrophilic EMR2 increase, whereas apoptosis induction disrupted it. Likely the damaged/necrotic cells/DAMPs, unavoidable during neutrophil culture, stimulate the neutrophilic EMR2 increase. In summary, the rapidly increased absolute number of neutrophils, especially present in very severely injured patients, together with upregulated neutrophilic EMR2, may expand our in vivo capacity to react to and finally clear damaged/necrotic cells/DAMPs after trauma.

A comprehensive model of the spatio-temporal stem cell and tissue organisation in the intestinal crypt.

We introduce a novel dynamic model of stem cell and tissue organisation in murine intestinal crypts. Integrating the molecular, cellular and tissue level of description, this model links a broad spectrum of experimental observations encompassing spatially confined cell proliferation, directed cell migration, multiple cell lineage decisions and clonal competition.Using computational simulations we demonstrate that the model is capable of quantitatively describing and predicting the dynamic behaviour of the intestinal tissue during steady state as well as after cell damage and following selective gain or loss of gene function manipulations affecting Wnt- and Notch-signalling. Our simulation results suggest that reversibility and flexibility of cellular decisions are key elements of robust tissue organisation of the intestine. We predict that the tissue should be able to fully recover after complete elimination of cellular subpopulations including subpopulations deemed to be functional stem cells. This challenges current views of tissue stem cell organisation.

To Detach, Migrate, Adhere, and Metastasize: CD97/ADGRE5 in Cancer.

Tumorigenesis is a multistep process, during which cells acquire a series of mutations that lead to unrestrained cell growth and proliferation, inhibition of cell differentiation, and evasion of cell death. Growing tumors stimulate angiogenesis, providing them with nutrients and oxygen. Ultimately, tumor cells invade the surrounding tissue and metastasize; a process responsible for about 90% of cancer-related deaths. Adhesion G protein-coupled receptors (aGPCRs) modulate the cellular processes closely related to tumor cell biology, such as adhesion and detachment, migration, polarity, and guidance. Soon after first being described, individual human aGPCRs were found to be involved in tumorigenesis. Twenty-five years ago, CD97/ADGRE5 was discovered to be induced in one of the most severe tumors, dedifferentiated anaplastic thyroid carcinoma. After decades of research, the time has come to review our knowledge of the presence and function of CD97 in cancer. In summary, CD97 is obviously induced or altered in many tumor entities; this has been shown consistently in nearly one hundred published studies. However, its high expression at circulating and tumor-infiltrating immune cells renders the systemic targeting of CD97 in tumors difficult.

Organoid Cultures In Silico: Tools or Toys?

The implementation of stem-cell-based organoid culture more than ten years ago started a development that created new avenues for diagnostic analyses and regenerative medicine. In parallel, computational modelling groups realized the potential of this culture system to support their theoretical approaches to study tissues in silico. These groups developed computational organoid models (COMs) that enabled testing consistency between cell biological data and developing theories of tissue self-organization. The models supported a mechanistic understanding of organoid growth and maturation and helped linking cell mechanics and tissue shape in general. What comes next? Can we use COMs as tools to complement the equipment of our biological and medical research? While these models already support experimental design, can they also quantitatively predict tissue behavior? Here, we review the current state of the art of COMs and discuss perspectives for their application.

The Adhesion G-Protein-Coupled Receptor GPR115/ADGRF4 Regulates Epidermal Differentiation and Associates with Cytoskeletal KRT1.

Among the 33 human adhesion G-protein-coupled receptors (aGPCRs), a unique subfamily of GPCRs, only ADGRF4, encoding GPR115, shows an obvious skin-dominated transcriptomic profile, but its expression and function in skin is largely unknown. Here, we report that GPR115 is present in a small subset of basal and in most suprabasal, noncornified keratinocytes of the stratified epidermis, supporting epidermal transcriptomic data. In psoriatic skin, characterized by hyperproliferation and delayed differentiation, the expression of GPR115 and KRT1/10, the fundamental suprabasal keratin dimer, is delayed. The deletion of ADGRF4 in HaCaT keratinocytes grown in an organotypic mode abrogates KRT1 and reduces keratinocyte stratification, indicating a role of GPR115 in epidermal differentiation. Unexpectedly, endogenous GPR115, which is not glycosylated and is likely not proteolytically processed, localizes intracellularly along KRT1/10-positive keratin filaments in a regular pattern. Our data demonstrate a hitherto unknown function of GPR115 in the regulation of epidermal differentiation and KRT1.

Glyphosate differentially affects the allergic immune response across generations in mice.

Exposure to environmental pollutants via food, particularly during the prenatal and early postnatal periods, has been linked to adverse effects on the immune system. Among these pollutants, the widely used pesticide glyphosate has been associated with endocrine disruption, autism, and cancer. Occupational high exposure to glyphosate has also been shown to influence immune function and exacerbate allergic asthma. However, there are no studies investigating the effect of a common low-dose glyphosate exposure on the allergic immune response - neither directly nor across generations. We therefore explored the impact of oral low-dose glyphosate exposure (0.5 and 50 mg/kg body weight/day) on airway inflammation in dams (F0) and the offspring (F1 and F2 generations) using a murine multi-generational asthma model. While exposure to 50 mg/kg glyphosate induced a mild eosinophilic infiltration in the bronchoalveolar lavage and TH2 cytokine production in the dams, the F1 offspring developed a reduced immune response after maternal exposure to 0.5 mg/kg glyphosate. In particular, decreased lung inflammation, HDM-specific IgE levels, and asthma-relevant cytokine production were primarily observed in the female F1 offspring. However, not only the TH2 cytokines IL-13 and IL-5 but also the TH17 cytokine IL-17 and TH1 cytokine IFN-γ were reduced indicating a more general immunosuppressive function. Notably, the dampened immune response was no longer observed in the female F2 generation. Furthermore, female F1 offspring showed an increased abundance of bacteria in the gut, which have been associated with probiotic-mediated reduced allergic immune responses. Our results suggest a potential immunosuppressive effect of low-dose maternal glyphosate exposure in the F1 offspring that might be mediated by an altered microbiota composition. Further studies are needed to explore if this type of immune response modulation might also be associated with impairments in immune defense upon infectious diseases or even cancer pathology.

CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia.

The heterogeneous response of acute myeloid leukemia (AML) to current anti-leukemic therapies is only partially explained by mutational heterogeneity. We previously identified GPR56 as a surface marker associated with poor outcome across genetic groups, which characterizes two leukemia stem cell (LSC)-enriched compartments with different self-renewal capacities. How these compartments self-renew remained unclear. Here, we show that GPR56+ LSC compartments are promoted in a complex network involving epithelial-to-mesenchymal transition (EMT) regulators besides Rho, Wnt, and Hedgehog (Hh) signaling. Unexpectedly, Wnt pathway inhibition increased the more immature, slowly cycling GPR56+ CD34+ fraction and Hh/EMT gene expression, while Wnt activation caused opposite effects. Our data suggest that the crucial role of GPR56 lies in its ability to co-activate these opposing signals, thus ensuring the constant supply of both LSC subsets. We show that CDK7 inhibitors suppress both LSC-enriched subsets in vivo and synergize with the Bcl-2 inhibitor venetoclax. Our data establish reciprocal transition between LSC compartments as a novel concept underlying the poor outcome in GPR56high AML and propose combined CDK7 and Bcl-2 inhibition as LSC-directed therapy in this disease.

Calcium signaling of thyrocytes is modulated by TSH through calcium binding protein expression.

TSH is an important stimulus to maintain thyroid epithelial differentiation. Impairment of TSH signal transduction can cause thyroid pathologies such as hot nodules, goiter and hyperthyroidism. In a gene expression study in Fischer rat thyroid cells (FRTL-5) using cDNA microarrays we found a TSH-dependent regulation of several calcium binding proteins, S100A4, S100A6 and annexin A6. Expression of these genes in FRTL-5 and regulation by TSH was confirmed with LightCycler qPCR and Western blotting. The differential expression of S100A4 was confirmed for cultured primary human thyrocytes. Calcium-imaging experiments showed that prestimulation with TSH attenuates ATP-elicited P2Y-mediated calcium signaling. Experiments with thapsigargin, TSH and calcium-free perfusion excluded an involvement of other purinergic receptors or an involvement of SERCA regulation. Instead, we find a correlation between S100A4 expression and the effects of TSH on calcium signaling. Overexpression of S100A4 in FRTL-5 and shRNA-mediated knockdown of S100A4 in follicular thyroid cancer cells (FTC133) confirm the ability of S100A4 to attenuate calcium signals. Under repeated stimulations with ATP the calcium retention of these cells is also modulated by S100A4, suggesting a role of S100A4 as calcium buffering protein. As a biological consequence of S100A4 overexpression we detected reduced ATP-stimulated cFos induction. Taken together, the results suggest that S100A4 and other calcium binding proteins are part of a signaling network connecting TSH signaling to calcium-mediated events which play a role in thyroid physiology like H2O2 production or even thyroid cancer.