YY1 DNA binding and interaction with YAF2 is essential for Polycomb recruitment.

Polycomb Group (PcG) proteins are crucial for epigenetic inheritance of cell identity and are functionally conserved from Drosophila to humans. PcG proteins regulate expression of homeotic genes and are essential for axial body patterning during development. Earlier we showed that transcription factor YY1 functions as a PcG protein. YY1 also physically interacts with YAF2, a homolog of RYBP. Here we characterize the mechanism and physiologic relevance of this interaction. We found phenotypic and biochemical correction of dRYBP mutant flies by mouse YAF2 demonstrating functional conservation across species. Further biochemical analysis revealed that YAF2 bridges interaction between YY1 and the PRC1 complex. ChIP assays in HeLa cells showed that YAF2 is responsible for PcG recruitment to DNA, which is mediated by YY1 DNA binding. Knock-down of YY1 abrogated PcG recruitment, which was not compensated by exogenous YAF2 demonstrating that YY1 DNA binding is a priori necessary for Polycomb assembly on chromatin. Finally, we found that although YAF2 and RYBP regulate a similar number of Polycomb target genes, there are very few genes that are regulated by both implying functional distinction between the two proteins. We present a model of YAF2-dependent and independent PcG DNA recruitment by YY1.

Cultivated Meat from Aligned Muscle Layers and Adipose Layers Formed from Glutenin Films.

Cultivated meat production is a promising technology to generate meat while reducing the reliance on traditional animal farming. Biomaterial scaffolds are critical components in cultivated meat production, enabling cell adhesion, proliferation, differentiation, and orientation. In the present work, naturally derived glutenin was fabricated into films with and without surface patterning and in the absence of toxic cross-linking or stabilizing agents for cell culture related to cultivated meat goals. The films were stable in culture media for at least 28 days, and the surface patterns induced cell alignment and guided myoblast organization (C2C12s) and served as a substrate for 3T3-L1 adipose cells. The films supported adhesion, proliferation, and differentiation with mass balance considerations (films, cells, and matrix production). Freeze-thaw cycles were applied to remove cells from glutenin films and monitor changes in glutenin mass with respect to culture duration. Extracellular matrix (ECM) extraction was utilized to quantify matrix deposition and changes in the original biomaterial mass over time during cell cultivation. Glutenin films with C2C12s showed mass increases with time due to cell growth and new collagen-based ECM expression during proliferation and differentiation. All mass balances were compared among cell and noncell systems as controls, along with gelatin control films, with time-dependent changes in the relative content of film, matrix deposition, and cell biomass. These data provide a foundation for cell/biomaterial/matrix ratios related to time in culture as well as nutritional and textural features.

Edible films for cultivated meat production.

Biomaterial scaffolds are critical components in cultivated meat production for enabling cell adhesion, proliferation, differentiation and orientation. Currently, there is limited information on the fabrication of edible/biodegradable scaffolds for cultivated meat applications. In the present work, several abundant, naturally derived biomaterials (gelatin, soy, glutenin, zein, cellulose, alginate, konjac, chitosan) were fabricated into films without toxic cross-linking or stabilizing agents. These films were investigated for support of the adhesion, proliferation and differentiation of murine and bovine myoblasts. These biomaterials supported cell viability, and the protein-based films showed better cell adhesion than the polysaccharide-based films. Surface patterns induced cell alignment and guided myoblast differentiation and organization on the glutenin and zein films. The mechanical properties of the protein films were also assessed and suggested that a range of properties can be achieved to meet food-related goals. Overall, based on adherence, proliferation, differentiation, mechanics, and material availability, protein-based films, particularly glutenin and zein, showed the most promise for cultivated meat applications. Ultimately, this work presents a comparison of suitable biomaterials for cultivated meat applications and suggests future efforts to optimize scaffolds for efficacy and cost.

Bioelectric Properties of Myogenic Progenitor Cells.

Modern stem cell research has mainly focused on protein expression and transcriptional networks. However, transmembrane voltage gradients generated by ion channels and transporters have demonstrated to be powerful regulators of cellular processes. These physiological cues exert influence on cell behaviors ranging from differentiation and proliferation to migration and polarity. Bioelectric signaling is a fundamental element of living systems and an untapped reservoir for new discoveries. Dissecting these mechanisms will allow for novel methods of controlling cell fate and open up new opportunities in biomedicine. This review focuses on the role of ion channels and the resting membrane potential in the proliferation and differentiation of skeletal muscle progenitor cells. In addition, findings relevant to this topic are presented and potential implications for tissue engineering and regenerative medicine are discussed.

PPT1 Promotes Tumor Growth and Is the Molecular Target of Chloroquine Derivatives in Cancer.

Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy significantly better than hydroxychloroquine (HCQ). Using an in situ photoaffinity pulldown strategy, we identified palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in significant impairment of tumor growth similar to that observed with DC661. Elevated expression of PPT1 in tumors correlates with poor survival in patients in a variety of cancers. Thus, PPT1 represents a new target in cancer that can be inhibited with CQ derivatives. SIGNIFICANCE: This study identifies PPT1 as the previously unknown lysosomal molecular target of monomeric and dimeric CQ derivatives. Genetic suppression of PPT1 impairs tumor growth, and PPT1 levels are elevated in cancer and associated with poor survival. These findings provide a strong rationale for targeting PPT1 in cancer. This article is highlighted in the In This Issue feature, p. 151.

ER Translocation of the MAPK Pathway Drives Therapy Resistance in BRAF-Mutant Melanoma.

Resistance to BRAF and MEK inhibitors (BRAFi + MEKi) in BRAF-mutant tumors occurs through heterogeneous mechanisms, including ERK reactivation and autophagy. Little is known about the mechanisms by which ERK reactivation or autophagy is induced by BRAFi + MEKi. Here, we report that in BRAF-mutant melanoma cells, BRAFi + MEKi induced SEC61-dependent endoplasmic reticulum (ER) translocation of the MAPK pathway via GRP78 and KSR2. Inhibition of ER translocation prevented ERK reactivation and autophagy. Following ER translocation, ERK exited the ER and was rephosphorylated by PERK. Reactivated ERK phosphorylated ATF4, which activated cytoprotective autophagy. Upregulation of GRP78 and phosphorylation of ATF4 were detected in tumors of patients resistant to BRAFi + MEKi. ER translocation of the MAPK pathway was demonstrated in therapy-resistant patient-derived xenografts. Expression of a dominant-negative ATF4 mutant conferred sensitivity to BRAFi + MEKi in vivo. This mechanism reconciles two major targeted therapy resistance pathways and identifies druggable targets, whose inhibition would likely enhance the response to BRAFi + MEKi. SIGNIFICANCE: ERK reactivation and autophagy are considered distinct resistance pathways to BRAF + MEK inhibition (BRAFi + MEKi) in BRAF V600E cancers. Here, we report BRAFi + MEKi-induced ER translocation of the MAPK pathway is necessary for ERK reactivation, which drives autophagy. The ER translocation mechanism is a major druggable driver of resistance to targeted therapy.This article is highlighted in the In This Issue feature, p. 305.

Lysosomal Biology in Cancer.

Cells depend on the lysosome for sequestration and degradation of macromolecules in order to maintain metabolic homeostasis. These membrane-enclosed organelles can receive intracellular and extracellular cargo through endocytosis, phagocytosis, and autophagy. Lysosomes establish acidic environments to activate enzymes that are able to break down biomolecules engulfed through these various pathways. Recent advances in methods to study the lysosome have allowed the discovery of extended roles for the lysosome in various diseases, including cancer, making it an attractive and targetable node for therapeutic intervention. This review focuses on key aspects of lysosomal biology in the context of cancer and how these properties can be exploited for the development of new therapeutic strategies. This will provide a contextual framework for how advances in methodology could be applied in future translational research.

A Unified Approach to Targeting the Lysosome's Degradative and Growth Signaling Roles.

Lysosomes serve dual roles in cancer metabolism, executing catabolic programs (i.e., autophagy and macropinocytosis) while promoting mTORC1-dependent anabolism. Antimalarial compounds such as chloroquine or quinacrine have been used as lysosomal inhibitors, but fail to inhibit mTOR signaling. Further, the molecular target of these agents has not been identified. We report a screen of novel dimeric antimalarials that identifies dimeric quinacrines (DQ) as potent anticancer compounds, which concurrently inhibit mTOR and autophagy. Central nitrogen methylation of the DQ linker enhances lysosomal localization and potency. An in situ photoaffinity pulldown identified palmitoyl-protein thioesterase 1 (PPT1) as the molecular target of DQ661. PPT1 inhibition concurrently impairs mTOR and lysosomal catabolism through the rapid accumulation of palmitoylated proteins. DQ661 inhibits the in vivo tumor growth of melanoma, pancreatic cancer, and colorectal cancer mouse models and can be safely combined with chemotherapy. Thus, lysosome-directed PPT1 inhibitors represent a new approach to concurrently targeting mTORC1 and lysosomal catabolism in cancer.Significance: This study identifies chemical features of dimeric compounds that increase their lysosomal specificity, and a new molecular target for these compounds, reclassifying these compounds as targeted therapies. Targeting PPT1 blocks mTOR signaling in a manner distinct from catalytic inhibitors, while concurrently inhibiting autophagy, thereby providing a new strategy for cancer therapy. Cancer Discov; 7(11); 1266-83. ©2017 AACR.See related commentary by Towers and Thorburn, p. 1218This article is highlighted in the In This Issue feature, p. 1201.

Sustained Depolarization of the Resting Membrane Potential Regulates Muscle Progenitor Cell Growth and Maintains Stem Cell Properties In Vitro.

It is important to maintain the myogenic properties of muscle progenitor cells (MPCs) during in vitro expansion for stem cell therapies and tissue engineering applications. Controlling cell fate for biomedical interventions will require insight on all aspects that influence cellular properties. The resting membrane potential (Vmem) has proven to be a key parameter involved in cell proliferation, migration and differentiation. This current work is focused on elucidating the impact of sustained depolarization on MPC growth and differentiation in vitro. Cultures were treated with either potassium gluconate or the sodium-potassium pump blocker ouabain and evaluated for proliferation, DNA content using propidum iodide staining, and differentiation. Cell proliferation measurements showed a modest stimulatory effect at certain concentration ranges for each agent, but higher concentrations of potassium gluconate strongly inhibited growth in a dose dependent manner. Cell cycle analysis with flow cytometry demonstrated an increase in the number of cells in S phase, but increasing concentrations of potassium gluconate arrested cells at G1. Immunostaining, Western blot analysis and light microscopy revealed that potassium gluconate exposure delayed cell fusion and maintained a higher population of cells expressing the muscle stem cell marker Pax7. The impairment on cell fusion was transient and myotube formation recovered after the treatments were removed. Taken together, this work suggests that transmembrane voltage gradients can be used as a powerful regulator of MPC properties in vitro. Examination of how these physiological parameters modulate cell behavior will reveal a new set of tools that can be capitalized on in tissue engineering and regenerative medicine.

An in vitro culture system that supports robust expansion and maintenance of in vivo engraftment capabilities for myogenic progenitor cells from adult mice.

Muscle cell therapy and tissue engineering require large numbers of functional muscle precursor/progenitor cells (MPCs), making the in vitro expansion of MPCs a critical step for these applications. The cells must maintain their myogenic properties upon robust expansion, especially for cellular therapy applications, in order to achieve efficacious treatment. A major obstacle associated with MPCs expansion is the loss of "stemness," or regenerative capacity, of freshly isolated cells, presumably due to the absence of the native cellular niches. In the current study, we developed an in vitro system that allowed for long-term culture and massive expansion of murine MPCs (mMPCs) with the preservation of myogenic regeneration capabilities. Long term in vitro expanded mMPC expressed the myogenic stem cell markers Pax3 and Pax7 and formed spontaneously contracting myotubes. Furthermore, expanded mMPC injected into the tibialis anterior muscle of nude mice engrafted and formed myofibers. Collectively, the method developed in this study can be potentially adapted for the expansion of human MPCs to high enough numbers for treatment of muscle injuries in human patients.