Characterization of mitochondrial health from human peripheral blood mononuclear cells to cerebral organoids derived from induced pluripotent stem cells.

Mitochondrial health plays a crucial role in human brain development and diseases. However, the evaluation of mitochondrial health in the brain is not incorporated into clinical practice due to ethical and logistical concerns. As a result, the development of targeted mitochondrial therapeutics remains a significant challenge due to the lack of appropriate patient-derived brain tissues. To address these unmet needs, we developed cerebral organoids (COs) from induced pluripotent stem cells (iPSCs) derived from human peripheral blood mononuclear cells (PBMCs) and monitored mitochondrial health from the primary, reprogrammed and differentiated stages. Our results show preserved mitochondrial genetics, function and treatment responses across PBMCs to iPSCs to COs, and measurable neuronal activity in the COs. We expect our approach will serve as a model for more widespread evaluation of mitochondrial health relevant to a wide range of human diseases using readily accessible patient peripheral (PBMCs) and stem-cell derived brain tissue samples.

Cyproheptadine displays preclinical activity in myeloma and leukemia.

D-cyclins are regulators of cell division that act in a complex with cyclin-dependent kinases to commit cells to a program of DNA replication. D-cyclins are overexpressed in many tumors, including multiple myeloma and leukemia, and contribute to disease progression and chemoresistance. To better understand the role and impact of D-cyclins in hematologic malignancies, we conducted a high throughput screen for inhibitors of the cyclin D2 promoter and identified the drug cyproheptadine. In myeloma and leukemia cells, cyproheptadine decreased expression of cyclins D1, D2, and D3 and arrested these cells in the G(0)/G(1) phase. After D-cyclin suppression, cyproheptadine induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of myeloma and leukemia, cyproheptadine inhibited tumor growth without significant toxicity. Cyproheptadine-induced apoptosis was preceded by activation of the mitochondrial pathway of caspase activation and was independent of the drug's known activity as an H1 histamine and serotonin receptor antagonist. Thus, cyproheptadine represents a lead for a novel therapeutic agent for the treatment of malignancy. Because the drug is well tolerated and already approved in multiple countries for clinical use as an antihistamine and appetite stimulant, it could be moved directly into clinical trials for cancer.

Functional nonsynonymous single nucleotide polymorphisms from the TGF-beta protein interaction network.

Protein complexes mediated by protein-protein interactions are essential for many cellular functions. Transforming growth factor (TGF)-beta signaling involves a cascade of protein-protein interactions and malfunctioning of this pathway has been implicated in human diseases. Using an in silico approach, we analyzed the naturally occurring human genetic variations from the proteins involved in the TGF-beta signaling (10 TGF-beta proteins and 242 other proteins interacting with them) to identify the ones that have potential biological consequences. All proteins were searched in the dbSNP database for the presence of nonsynonymous single nucleotide polymorphisms (nsSNPs). A total of 118 validated nsSNPs from 63 proteins were retrieved and analyzed in terms of 1) evolutionary conservation status, 2) being located in a functional protein domain or motif, and 3) altering putative protein motif or phosphorylation sites. Our results indicated the presence of 31 nsSNPs that occurred at evolutionarily conserved residues, 37 nsSNPs were located in protein domains, motifs, or repeats, and 46 nsSNPs were predicted to either create or abolish putative protein motifs or phosphorylation sites. We undertook this study to analyze the human genetic variations that can affect the protein function and the TGF-beta signaling. The nsSNPs reported in here can be characterized by experimental approaches to elucidate their exact biological roles and whether they are related to human disease.

alpha2HS-glycoprotein, an antagonist of transforming growth factor beta in vivo, inhibits intestinal tumor progression.

Transforming growth factor (TGF)-beta1 is associated with tumor progression and resistance to chemotherapy in established cancers, as well as host immune suppression. Here, we show that the serum glycoprotein alpha2-HS-glycoprotein (AHSG) blocks TGF-beta1 binding to cell surface receptors, suppresses TGF-beta signal transduction, and inhibits TGF-beta-induced epithelial-mesenchymal transition, suggesting that AHSG may play a role in tumor progression. In 66 consecutive sporadic human colorectal cancer specimens, we observed a 3-fold depletion of ASHG in tumor compared with normal tissue, whereas levels of other abundant plasma proteins, albumin and transferrin, were equivalent. Using the Multiple intestinal neoplasia/+ (Min/+) mouse model of intestinal tumorigenesis, we found twice as many intestinal polyps overall, twice as many large polyps (>3 mm diameter), and more progression to invasive adenocarcinoma in Min/+ Ahsg-/- mice than in littermates expressing Ahsg. Phosphorylated Smad2 was more abundant in the intestinal mucosa and tumors of Min/+ mice lacking Ahsg, demonstrating increased TGF-beta signaling in vivo. Furthermore, TGF-beta-mediated suppression of immune cell function was exaggerated in Ahsg-/- animals, as shown by inhibition of macrophage activation and reduction in 12-O-tetradecanoylphorbol 13-acetate-induced cutaneous inflammation. Reconstitution of Ahsg-/- mice with bovine Ahsg suppressed endogenous TGF-beta-dependent signaling to wild-type levels, suggesting that therapeutic enhancement of AHSG levels may benefit patients whose tumors are driven by TGF-beta.

The actin-binding protein EPS8 binds VE-cadherin and modulates YAP localization and signaling.

Vascular endothelial (VE)-cadherin transfers intracellular signals contributing to vascular hemostasis. Signaling through VE-cadherin requires association and activity of different intracellular partners. Yes-associated protein (YAP)/TAZ transcriptional cofactors are important regulators of cell growth and organ size. We show that EPS8, a signaling adapter regulating actin dynamics, is a novel partner of VE-cadherin and is able to modulate YAP activity. By biochemical and imaging approaches, we demonstrate that EPS8 associates with the VE-cadherin complex of remodeling junctions promoting YAP translocation to the nucleus and transcriptional activation. Conversely, in stabilized junctions, 14-3-3-YAP associates with the VE-cadherin complex, whereas Eps8 is excluded. Junctional association of YAP inhibits nuclear translocation and inactivates its transcriptional activity both in vitro and in vivo in Eps8-null mice. The absence of Eps8 also increases vascular permeability in vivo, but did not induce other major vascular defects. Collectively, we identified novel components of the adherens junction complex, and we introduce a novel molecular mechanism through which the VE-cadherin complex controls YAP transcriptional activity.

Wnt inhibitor screen reveals iron dependence of ß-catenin signaling in cancers.

Excessive signaling from the Wnt pathway is associated with numerous human cancers. Using a high throughput screen designed to detect inhibitors of Wnt/ß-catenin signaling, we identified a series of acyl hydrazones that act downstream of the ß-catenin destruction complex to inhibit both Wnt-induced and cancer-associated constitutive Wnt signaling via destabilization of ß-catenin. We found that these acyl hydrazones bind iron in vitro and in intact cells and that chelating activity is required to abrogate Wnt signaling and block the growth of colorectal cancer cell lines with constitutive Wnt signaling. In addition, we found that multiple iron chelators, desferrioxamine, deferasirox, and ciclopirox olamine similarly blocked Wnt signaling and cell growth. Moreover, in patients with AML administered ciclopirox olamine, we observed decreased expression of the Wnt target gene AXIN2 in leukemic cells. The novel class of acyl hydrazones would thus be prime candidates for further development as chemotherapeutic agents. Taken together, our results reveal a critical requirement for iron in Wnt signaling and they show that iron chelation serves as an effective mechanism to inhibit Wnt signaling in humans.