Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types.

Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.

Apc-mutant cells act as supercompetitors in intestinal tumour initiation.

A delicate equilibrium of WNT agonists and antagonists in the intestinal stem cell (ISC) niche is critical to maintaining the ISC compartment, as it accommodates the rapid renewal of the gut lining. Disruption of this balance by mutations in the tumour suppressor gene APC, which are found in approximately 80% of all human colon cancers, leads to unrestrained activation of the WNT pathway1,2. It has previously been established that Apc-mutant cells have a competitive advantage over wild-type ISCs3. Consequently, Apc-mutant ISCs frequently outcompete all wild-type stem cells within a crypt, thereby reaching clonal fixation in the tissue and initiating cancer formation. However, whether the increased relative fitness of Apc-mutant ISCs involves only cell-intrinsic features or whether Apc mutants are actively involved in the elimination of their wild-type neighbours remains unresolved. Here we show that Apc-mutant ISCs function as bona fide supercompetitors by secreting WNT antagonists, thereby inducing differentiation of neighbouring wild-type ISCs. Lithium chloride prevented the expansion of Apc-mutant clones and the formation of adenomas by rendering wild-type ISCs insensitive to WNT antagonists through downstream activation of WNT by inhibition of GSK3ß. Our work suggests that boosting the fitness of healthy cells to limit the expansion of pre-malignant clones may be a powerful strategy to limit the formation of cancers in high-risk individuals.

The Wnt Antagonist Dickkopf-1 Promotes Pathological Type 2 Cell-Mediated Inflammation.

Exposure to a plethora of environmental challenges commonly triggers pathological type 2 cell-mediated inflammation. Here we report the pathological role of the Wnt antagonist Dickkopf-1 (Dkk-1) upon allergen challenge or non-healing parasitic infection. The increased circulating amounts of Dkk-1 polarized T cells to T helper 2 (Th2) cells, stimulating a marked simultaneous induction of the transcription factors c-Maf and Gata-3, mediated by the kinases p38 MAPK and SGK-1, resulting in Th2 cell cytokine production. Circulating Dkk-1 was primarily from platelets, and the increase of Dkk-1 resulted in formation of leukocyte-platelet aggregates (LPA) that facilitated leukocyte infiltration to the affected tissue. Functional inhibition of Dkk-1 impaired Th2 cell cytokine production and leukocyte infiltration, protecting mice from house dust mite (HDM)-induced asthma or Leishmania major infection. These results highlight that Dkk-1 from thrombocytes is an important regulator of leukocyte infiltration and polarization of immune responses in pathological type 2 cell-mediated inflammation.

Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium.

A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche1,2. Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor α (PPAR-α)3, and lowered PPAR-α activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues.

A slow-cycling LGR5 tumour population mediates basal cell carcinoma relapse after therapy.

Basal cell carcinoma (BCC) is the most frequent cancer in humans and results from constitutive activation of the Hedgehog pathway1. Several Smoothened inhibitors are used to treat Hedgehog-mediated malignancies, including BCC and medulloblastoma2. Vismodegib, a Smoothened inhibitor, leads to BCC shrinkage in the majority of patients with BCC3, but the mechanism by which it mediates BCC regression is unknown. Here we used two genetically engineered mouse models of BCC4 to investigate the mechanisms by which inhibition of Smoothened mediates tumour regression. We found that vismodegib mediates BCC regression by inhibiting a hair follicle-like fate and promoting the differentiation of tumour cells. However, a small population of tumour cells persists and is responsible for tumour relapse following treatment discontinuation, mimicking the situation found in humans5. In both mouse and human BCC, this persisting, slow-cycling tumour population expresses LGR5 and is characterized by active Wnt signalling. Combining Lgr5 lineage ablation or inhibition of Wnt signalling with vismodegib treatment leads to eradication of BCC. Our results show that vismodegib induces tumour regression by promoting tumour differentiation, and demonstrates that the synergy between Wnt and Smoothened inhibitors is a clinically relevant strategy for overcoming tumour relapse in BCC.

Wnt signaling in the niche.

There is much interest in understanding the signals in the bone marrow niche that keep hematopoietic stem cells (HSCs) in a quiescent state. In the current issue of Cell Stem Cell, Fleming et al. (2008) report that blocking Wnt signaling in the niche increases the number of proliferating HSCs and reduces their ability to reconstitute the hematopoietic system of irradiated recipient mice. These findings show that Wnt/beta-catenin activity is crucial for the maintenance of HSC quiescence in the bone marrow niche.

Decreased Serum Wnt Antagonist Levels in Patients With Active Acromegaly.

OBJECTIVE: The Wnt signaling pathway is an important modulator of bone metabolism. This study aims to clarify the changes in Wnt antagonists in active and biochemically controlled acromegalic patients. METHODS: We recruited 77 patients recently diagnosed with acromegaly. Of those, 41 patients with complete follow-up data were included. Thirty healthy patients matched for age, sex, and body mass index served as controls. At baseline and posttreatment, Wnt antagonists (sclerostin [SOST], dickkopf-related protein 1 [DKK-1], and Wnt inhibitory factor 1 [WIF-1]), bone turnover markers (osteocalcin, procollagen type 1 N-terminal propeptide [P1NP], and C-terminal telopeptide of type 1 collagen [CTX]) and the bone remodeling index were investigated. RESULTS: Acromegalic patients had higher serum osteocalcin, P1NP, and CTX and a higher bone remodeling index than controls (P < .01). Serum SOST, DKK-1, and WIF-1 levels were significantly decreased in patients compared to controls (all P < .01). Serum SOST and WIF-1 levels were negatively correlated with growth hormone levels; SOST levels were positively correlated with WIF-1. After treatment, serum bone turnover markers and the bone remodeling index decreased, while SOST and WIF-1 significantly increased (P < .05). DKK-1 levels did not change compared to baseline (P > .05). In biochemically controlled patients, SOST and WIF-1 levels and bone turnover markers were restored and did not differ from those of the control participants (all P > .05). CONCLUSION: Patients with active acromegaly exhibited significantly decreased Wnt antagonist levels. The reduction in Wnt antagonists is a compensatory mechanism to counteract increased bone fragility in active acromegaly.

Wnt/ß-catenin inhibitor pyrivinium attenuates cisplatin-induced acute kidney injury by possibly reducing platinum uptake and accumulation mediated by reduced organic cation transporter-2 expressions.

Aberrant activation of Wnt/ß-catenin induces renal dysfunction by initiating pro-apoptotic cascades, fibrosis, oxidative and inflammatory burden. This study tested the therapeutic effects of Wnt/ß-catenin inhibitor pyrvinium against cisplatin-induced acute kidney injury (AKI) in rats. Cisplatin was administered at a single dose of 5 mg/kg (i.p.) and renal cisplatin accumulation and uptake in cortical slices were determined after the fifth day by atomic absorption spectroscopy. Levels of pro-inflammatory cytokines were checked by ELISA, and organic cation transporter-2 (OCT-2) transcription and expression in renal tissue were evaluated by RT-PCR and immunohistochemical technique. Cisplatin administration produced renal dysfunction manifested as increase in serum creatinine, blood urea nitrogen, proteinuria, reduced clearance and electrolyte imbalance. Oxidative stress indices, pro-inflammatory cytokines, fibronectin, and caspase-3 activity were elevated in cisplatin-challenged rats. Moreover, increased renal OCT-2 transcription and immunostaining were detected in cisplatin kidneys which resulted in platinum accumulation. Additional docking studies depicted strong interaction between the ß-catenin and OCT-2 protein. These manifestations induced mitochondrial dysfunction, histological damage and fibrosis. Notably, Wnt/ß-catenin inhibitor pyrvinium (60 µg/kg; p.o.) treatment reduced the renal OCT-2 gene transcription causing a decline in platinum levels. Thus, the present study concludes that Wnt/ß-catenin inhibition attenuates cisplatin-induced AKI in rats, partly by down-regulating OCT-2 expression.

α-Klotho released from HK-2 cells inhibits osteogenic differentiation of renal interstitial fibroblasts by inactivating the Wnt-ß-catenin pathway.

Randall's plaques (RP) are well established as precursor lesions of idiopathic calcium oxalate (CaOx) stones, and the process of biomineralization driven by osteogenic-like cells has been highlighted in RP formation, but the mechanism is poorly understood. Given the inhibitory role of α-Klotho (KL), an aging suppressor protein with high expression in kidneys, in ectopic calcification and the close association between KL gene polymorphisms and urolithiasis susceptibility, we determined the potential role of KL in RP formation. This study found that both soluble KL (s-KL) and transmembrane KL (m-KL) were downregulated, and that s-KL but not m-KL was inversely correlated with upregulation of osteogenic markers in RP tissues. Additionally, s-KL expression was markedly suppressed in human renal interstitial fibroblasts (hRIFs) and slightly suppressed in HK-2 cells after osteogenic induction, intriguingly, which was echoed to the greater osteogenic capability of hRIFs than HK-2 cells. Further investigations showed the inhibitory effect of s-KL on hRIF osteogenic differentiation in vitro and in vivo. Moreover, coculture with recombinant human KL (r-KL) or HK-2 cells suppressed osteogenic differentiation of hRIFs, and this effect was abolished by coculture with KL-silenced HK-2 cells or the ß-catenin agonist SKL2001. Mechanistically, s-KL inactivated the Wnt-ß-catenin pathway by directly binding to Wnt2 and upregulating SFRP1. Further investigations identified activation of the Wnt-ß-catenin pathway and downregulation of SFRP1 and DKK1 in RP tissues. In summary, this study identified s-KL deficiency as a pathological feature of RP and revealed that s-KL released from HK-2 cells inhibited osteogenic differentiation of hRIFs by inactivating the Wnt-ß-catenin pathway, not only providing in-depth insight into the role of s-KL in renal interstitial biomineralization but also shedding new light on the interaction of renal tubular epithelial cells with interstitial cells to clarify RP formation.

The multifaceted NF-kB: are there still prospects of its inhibition for clinical intervention in pediatric central nervous system tumors?

Despite advances in the understanding of the molecular mechanisms underlying the basic biology and pathogenesis of pediatric central nervous system (CNS) malignancies, patients still have an extremely unfavorable prognosis. Over the years, a plethora of natural and synthetic compounds has emerged for the pharmacologic intervention of the NF-kB pathway, one of the most frequently dysregulated signaling cascades in human cancer with key roles in cell growth, survival, and therapy resistance. Here, we provide a review about the state-of-the-art concerning the dysregulation of this hub transcription factor in the most prevalent pediatric CNS tumors: glioma, medulloblastoma, and ependymoma. Moreover, we compile the available literature on the anti-proliferative effects of varied NF-kB inhibitors acting alone or in combination with other therapies in vitro, in vivo, and clinical trials. As the wealth of basic research data continues to accumulate, recognizing NF-kB as a therapeutic target may provide important insights to treat these diseases, hopefully contributing to increase cure rates and lower side effects related to therapy.

Dynamic Expression of Transient Receptor Potential Vanilloid-3 and Integrated Signaling with Growth Factor Pathways during Lung Epithelial Wound Repair following Wood Smoke Particle and Other Forms of Lung Cell Injury.

Prior studies revealed increased expression of the transient receptor potential vanilloid-3 (TRPV3) ion channel after wood smoke particulate matter (WSPM) treatment of human bronchial epithelial cells (HBECs). TRPV3 attenuated pathologic endoplasmic reticulum stress and cytotoxicity mediated by transient receptor potential ankyrin-1. Here, the basis for how TRPV3 expression is regulated by cell injury and the effects this has on HBEC physiology and WSPM-induced airway remodeling in mice was investigated. TRPV3 mRNA was rapidly increased in HBECs treated with WSPM and after monolayer damage caused by tryptic disruption, scratch wounding, and cell passaging. TRPV3 mRNA abundance varied with time, and stimulated expression occurred independent of new protein synthesis. Overexpression of TRPV3 in HBECs reduced cell migration and wound repair while enhancing cell adhesion. This phenotype correlated with disrupted mRNA expression of ligands of the epidermal growth factor, tumor growth factor-ß, and frizzled receptors. Accordingly, delayed wound repair by TRPV3 overexpressing cells was reversed by growth factor supplementation. In normal HBECs, TRPV3 upregulation was triggered by exogenous growth factor supplementation and was attenuated by inhibitors of growth factor receptor signaling. In mice, subacute oropharyngeal instillation with WSPM also promoted TRPV3 mRNA expression and epithelial remodeling, which was attenuated by TRPV3 antagonist pre- and cotreatment. This latter effect may be the consequence of antagonist-induced TRPV3 expression. These findings provide insights into the roles of TRPV3 in lung epithelial cells under basal and dynamic states, as well as highlight potential roles for TRPV3 ligands in modulating epithelial damage/repair. SIGNIFICANCE STATEMENT: Coordinated epithelial repair is essential for the maintenance of the airways, with deficiencies and exaggerated repair associated with adverse consequences to respiratory health. This study shows that TRPV3, an ion channel, is involved in coordinating repair through integrated repair signaling pathways, wherein TRPV3 expression is upregulated immediately after injury and returns to basal levels as cells complete the repair process. TRPV3 may be a novel target for understanding and/or treating conditions in which airway/lung epithelial repair is not properly orchestrated.

Dysregulation of Wnt/ß-catenin signaling by protein kinases in hepatocellular carcinoma and its therapeutic application.

Wnt/ß-catenin signaling is indispensable for many biological processes, including embryonic development, cell cycle, inflammation, and carcinogenesis. Aberrant activation of the Wnt/ß-catenin signaling can promote tumorigenicity and enhance metastatic potential in hepatocellular carcinoma (HCC). Targeting this pathway is a new opportunity for precise medicine for HCC. However, inhibiting Wnt/ß-catenin signaling alone is unlikely to significantly improve HCC patient outcome due to the lack of specific inhibitors and the complexity of this pathway. Combination with other therapies will be an important next step in improving the efficacy of Wnt/ß-catenin signaling inhibitors. Protein kinases play a key and evolutionarily conserved role in the Wnt/ß-catenin signaling and have become one of the most important drug targets in cancer. Targeting Wnt/ß-catenin signaling and its regulatory kinase together will be a promising HCC management strategy. In this review, we summarize the kinases that modulate the Wnt/ß-catenin signaling in HCC and briefly discuss their molecular mechanisms. Furthermore, we list some small molecules that target the kinases and may inhibit Wnt/ß-catenin signaling, to offer new perspectives for preclinical and clinical HCC studies.

Dkk1 exacerbates doxorubicin-induced cardiotoxicity by inhibiting the Wnt/ß-catenin signaling pathway.

The cancer clinical therapy of doxorubicin (Dox) treatment is limited by its life-threatening cardiotoxic effects. Dickkopf-1 (Dkk1), the founding and best-studied member of the Dkk family, functions as an antagonist of canonical Wnt/ß-catenin. Dkk1 is considered to play a broad role in a variety of biological processes, but its effects on Dox-induced cardiomyopathy are poorly understood. Here, we found that the level of Dkk1 was significantly increased in Dox-treated groups, and this increase exacerbated Dox-induced cardiomyocyte apoptosis and mitochondrial dysfunction. Overexpressing Dkk1 aggravated Dox-induced cardiotoxicity in H9C2 cells. Similar results were detected when adding active Dkk1 protein extracellularly. Conversely, adding specific antibody blocking extracellular Dkk1 attenuated the cardiotoxic response to Dox. Adenovirus encoding Dkk1 was transduced through intramyocardial injection and exacerbated Dox-induced cardiomyocyte apoptosis, mitochondrial damage and heart injury in vivo Furthermore, Wnt/ß-catenin signaling was inhibited during Dox-induced cardiotoxicity, and the re-activation of ß-catenin prevented the effect of overexpressed Dkk1 and Dox-induced cardiotoxicity. In conclusion, these results reveal the crucial role of the Dkk1-Wnt/ß-catenin signaling axis in the process of Dox-induced cardiotoxicity and provide novel insights into the potential mechanism of cardiomyopathy caused by clinical application of Dox.

Divergent early mesoderm specification underlies distinct head and trunk muscle programmes in vertebrates.

Head and trunk muscles have discrete embryological origins and are governed by distinct regulatory programmes. Whereas the developmental route of trunk muscles from mesoderm is well studied, that of head muscles is ill defined. Here, we show that, unlike the myogenic trunk paraxial mesoderm, head mesoderm development is independent of the T/Tbx6 network in mouse. We reveal that, in contrast to Wnt and FGF-driven trunk mesoderm, dual inhibition of Wnt/ß-catenin and Nodal specifies head mesoderm. Remarkably, the progenitors derived from embryonic stem cells by dual inhibition efficiently differentiate into cardiac and skeletal muscle cells. This twin potential is the defining feature of cardiopharyngeal mesoderm: the head subtype giving rise to heart and branchiomeric head muscles. Therefore, our findings provide compelling evidence that dual inhibition specifies head mesoderm and unravel the mechanism that diversifies head and trunk muscle programmes during early mesoderm fate commitment. Significantly, this is the first report of directed differentiation of pluripotent stem cells, without transgenes, into progenitors with muscle/heart dual potential. Ability to generate branchiomeric muscle in vitro could catalyse efforts in modelling myopathies that selectively involve head muscles.

Wnt antagonist DKK-1 levels in systemic sclerosis are lower in skin but not in blood and are regulated by microRNA33a-3p.

BACKGROUND: Systemic sclerosis is an autoimmune skin disease which is associated with inflammation and resulting skin fibrosis. Myofibroblasts are the key cell type associated with the fibrosis but how they are differentiated is not clear. DKK-1 is a Wnt antagonist that blocks Wnt-mediated fibrosis and is reduced in fibrotic conditions. Thus, DKK-1 is a clear negative regulator of fibrosis in systemic sclerosis and its regulation is unknown. The aim of this work is to determine the levels of DKK-1 in serum and tissues of SSc and its regulation. METHODS: Skin biopsies were taken from early diffuse systemic sclerosis patients and healthy controls and DKK-1 measured by ELISA; serum was also isolated and DKK-1 quantified. DKK-1 was also measured by qRT-PCR. MicroRNA33a-3p was measured by TaqMan PCR. miR mimics and controls were transfected into dermal fibroblasts. Bleomycin mouse model was employed and compared to vehicle control treated mice, and gene expression was employed for DKK-1 and various extracellular matrix genes. RESULTS: DKK-1 is reduced in SSc skin and fibroblasts but is not reduced in the circulation in patients. MicroRNA33a-3p regulates DKK-1 levels epigenetically and is significantly reduced in SSc cells and whole tissue. DKK-1 is also reduced in the bleomycin mouse model and pro-fibrotic genes elevated. CONCLUSION: DKK-1 is reduced in SSc cells and is regulated by miR33a-3p, and restoring DKK-1 levels through epigenetic means could be a therapeutic target in systemic sclerosis.

Optimizations of a novel fluorescence polarization-based high-throughput screening assay for ß-catenin/LEF1 interaction inhibitors.

Aberrant activation of the Wnt/ß-catenin signaling pathway is prominent in the development and metastasis of non-small cell lung cancer (NSCLC). Highly effective inhibition of this pathway highlights a therapeutic avenue against NSCLC. Moreover, ß-catenin/LEF1 interaction regulates ß-catenin nuclear transport as well as the transcriptions of the key oncogenes in Wnt/ß-catenin signaling pathway. Therefore, interruption of this interaction would be a promising therapeutic strategy for NSCLC metastasis. To date, no economical and rapid high-throughput screening (HTS) assay has been reported for the discovery of ß-catenin/LEF1 interaction inhibitors. In this study, we developed a novel fluorescence polarization (FP)-based HTS assay to identify ß-catenin/LEF1 interaction inhibitors. The FITC-LEF1 sequence, incubation time, temperature, and DMSO resistance were optimized, and then a high Z' factor of 0.77 was achieved. A pilot screening of a natural product library via this established FP screening assay identified sanguinarine analogues as potential ß-catenin/LEF1 interaction inhibitors. GST pull-down and surface plasmon resonance (SPR) assay demonstrated that ß-catenin/LEF1 interaction is a potential anticancer target of sanguinarine in vitro. This newly developed FP screening assay will be vital for the rapid discovery of novel Wnt inhibitors targeting ß-catenin/LEF1 interaction.

Manipulating the Wnt/ß-catenin signaling pathway to promote anti-tumor immune infiltration into the TME to sensitize ovarian cancer to ICB therapy.

OBJECTIVE: Immune checkpoint blockade (ICB) therapy shows limited efficacy in ovarian cancers due to the "cold" immune phenotype surrounding these tumors. Previous studies have shown that in ovarian cancer Wnt/ß-catenin pathway activation contributes to this immune phenotype. Here, we evaluated the anti-tumor and immune-enhancing properties of the Wnt inhibitor, CGX-1321, used alone or in combination with either DKN-01 or anti-PD-1 therapy, in pre-clinical ovarian cancer models. METHODS: The parental ID8 murine ovarian cancer model harboring a knock-out of p53 (ID8p53-/-) and MISIIR-Tag spontaneous ovarian cancer models were used to test the effects of CGX-1321 alone or in combination therapies on tumor burden and immune cell landscape in the tumor microenvironment (TME). Flow cytometry and NanoString analyses were used to characterize the changes in tumor-intrinsic signaling and immune-related profiles in the TME of ovarian cancer in response to treatments. RESULTS: CGX-1321 significantly reduced tumor burden and constrained tumor progression in the ID8p53-/- and MISIIR-Tag models. Furthermore, CGX-1321 increased infiltrating CD8+ T cells in the TME. Combining CGX-1321 with either DKN-01 or anti-PD-1 therapy also decreased tumor burden and increased CD8+ T cell infiltration in the omentum TME but did not do so to a greater extent that CGX-1321 monotherapy. CONCLUSIONS: CGX-1321 significantly reduced tumor burden and enhanced CD8+ T cell levels in ovarian cancer, nevertheless the addition of DKN-01 or anti-PD-1 therapies did not enhance these effects of CGX-1321. Further investigation is needed to determine if CGX-1321 + DKN-01 combination treatment sensitizes pre-clinical ovarian cancer to ICB therapy.

ZNRF3 functions in mammalian sex determination by inhibiting canonical WNT signaling.

Mammalian sex determination is controlled by the antagonistic interactions of two genetic pathways: The SRY-SOX9-FGF9 network promotes testis determination partly by opposing proovarian pathways, while RSPO1/WNT-ß-catenin/FOXL2 signals control ovary development by inhibiting SRY-SOX9-FGF9. The molecular basis of this mutual antagonism is unclear. Here we show that ZNRF3, a WNT signaling antagonist and direct target of RSPO1-mediated inhibition, is required for sex determination in mice. XY mice lacking ZNRF3 exhibit complete or partial gonadal sex reversal, or related defects. These abnormalities are associated with ectopic WNT/ß-catenin activity and reduced Sox9 expression during fetal sex determination. Using exome sequencing of individuals with 46,XY disorders of sex development, we identified three human ZNRF3 variants in very rare cases of XY female presentation. We tested two missense variants and show that these disrupt ZNRF3 activity in both human cell lines and zebrafish embryo assays. Our data identify a testis-determining function for ZNRF3 and indicate a mechanism of direct molecular interaction between two mutually antagonistic organogenetic pathways.

From Pathogenesis to Therapy in Knee Osteoarthritis: Bench-to-Bedside.

Osteoarthritis (OA) is currently the most widespread musculoskeletal condition and primarily affects weight-bearing joints such as the knees and hips. Importantly, knee OA remains a multifactorial whole-joint disease, the appearance and progression of which involves the alteration of articular cartilage as well as the synovium, subchondral bone, ligaments, and muscles through intricate pathomechanisms. Whereas it was initially depicted as a predominantly aging-related and mechanically driven condition given its clear association with old age, high body mass index (BMI), and joint malalignment, more recent research identified and described a plethora of further factors contributing to knee OA pathogenesis. However, the pathogenic intricacies between the molecular pathways involved in OA prompted the study of certain drugs for more than one therapeutic target (amelioration of cartilage and bone changes, and synovial inflammation). Most clinical studies regarding knee OA focus mainly on improvement in pain and joint function and thus do not provide sufficient evidence on the possible disease-modifying properties of the tested drugs. Currently, there is an unmet need for further research regarding OA pathogenesis as well as the introduction and exhaustive testing of potential disease-modifying pharmacotherapies in order to structure an effective treatment plan for these patients.

Management of Myeloma Bone Lesions.

Multiple myeloma (MM) is a B-cell neoplasm characterized by clonal plasma-cell proliferation. The survival and prognosis of this condition have been significantly improved by treatment with active anti-MM drugs such as bortezomib or lenalidomide. Further, the discovery of novel agents has recently paved the way for new areas of investigation. However, MM, including myeloma-related bone diseases, remains fatal. Bone disease or bone destruction in MM is a consequence of skeletal involvement with bone pain, spinal cord compression, and bone fracture resulting from osteolytic lesions. These consequences affect disease outcomes, including patients' quality of life and survival. Several studies have sought to better understand MM bone disease (MBD) through the classification of its molecular mechanisms, including osteoclast activation and osteoblast inhibition. Bisphosphonates and the receptor activator of the nuclear factor-kappa B (NF-κB) ligand (RANKL) inhibitor, denosumab, prevent skeletal-related events in MM. In addition, several other bone-targeting agents, including bone-anabolic drugs, are currently used in preclinical and early clinical evaluations. This review summarizes the current knowledge of the pathogenesis of MBD and discusses novel agents that appear very promising and will soon enter clinical development.