Dual inhibition of HSF1 and DYRK2 impedes cancer progression.

Preserving proteostasis is a major survival mechanism for cancer. Dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is a key oncogenic kinase that directly activates the transcription factor heat-shock factor 1 (HSF1) and the 26S proteasome. Targeting DYRK2 has proven to be a tractable strategy to target cancers sensitive to proteotoxic stress; however, the development of HSF1 inhibitors remains in its infancy. Importantly, multiple other kinases have been shown to redundantly activate HSF1 that promoted ideas to directly target HSF1. The eventual development of direct HSF1 inhibitor KRIBB11 suggests that the transcription factor is indeed a druggable target. The current study establishes that concurrent targeting of HSF1 and DYRK2 can indeed impede cancer by inducing apoptosis faster than individual targetting. Furthermore, targeting the DYRK2-HSF1 axis induces death in proteasome inhibitor-resistant cells and reduces triple-negative breast cancer (TNBC) burden in ectopic and orthotopic xenograft models. Together the data indicate that cotargeting of kinase DYRK2 and its substrate HSF1 could prove to be a beneficial strategy in perturbing neoplastic malignancies.

Hexosamine pathway activation improves memory but does not extend lifespan in mice.

Glucosamine feeding and genetic activation of the hexosamine biosynthetic pathway (HBP) have been linked to improved protein quality control and lifespan extension. However, as an energy sensor, the HBP has been implicated in tumor progression and diabetes. Given these opposing outcomes, it is imperative to explore the long-term effects of chronic HBP activation in mammals. Thus, we asked if HBP activation affects metabolism, coordination, memory, and survival in mice. N-acetyl-D-glucosamine (GlcNAc) supplementation in the drinking water had no adverse effect on weight in males but increased weight in young females. Glucose or insulin tolerance was not affected up to 20 months of age. Of note, we observed improved memory in young male mice supplemented with GlcNAc. Survival was not changed by GlcNAc treatment. To assess the effects of genetic HBP activation, we overexpressed the pathway's key enzyme GFAT1 and a constitutively activated mutant form in all mouse tissues. We detected elevated levels of the HBP product UDP-GlcNAc in mouse brains, but did not find any effects on behavior, memory, or survival. Together, while dietary GlcNAc supplementation did not extend survival in mice, it positively affected memory and is generally well tolerated.

The RATIOnal Role of Polyamines in Epidermal Differentiation.

Polyamines have been implicated in skin tumorigenesis; however, their role in epidermal homeostasis remains obscure. In a new article in the Journal of Investigative Dermatology, Rahim et al. (2021) report that keratinocyte differentiation requires a shift in polyamine ratios that is mediated by AMD1. Results suggest that targeting polyamine availability might be useful in the treatment of hyperproliferative skin disorders.

Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1.

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.

Small-molecule inhibitors of human mitochondrial DNA transcription.

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.

The E3 ubiquitin ligase UBR5 interacts with the H/ACA ribonucleoprotein complex and regulates ribosomal RNA biogenesis in embryonic stem cells.

UBR5 is an E3 ubiquitin ligase involved in distinct processes such as transcriptional regulation and development. UBR5 is highly upregulated in embryonic stem cells (ESCs), whereas its expression decreases with differentiation, suggesting a role for UBR5 in ESC function. However, little is known about how UBR5 regulates ESC identity. Here, we define the protein interactome of UBR5 in ESCs and find interactions with distinct components of the H/ACA ribonucleoprotein complex, which is required for proper maturation of ribosomal RNA (rRNA). Notably, loss of UBR5 induces an abnormal accumulation of rRNA processing intermediates, resulting in diminished ribosomal levels. Consequently, lack of UBR5 triggers an increase in p53 levels and a concomitant decrease in cellular proliferation rates. Thus, our results indicate a link between UBR5 and rRNA maturation.

Unbiased compound-protein interface mapping and prediction of chemoresistance loci through forward genetics in haploid stem cells.

Forward genetic screens in haploid mammalian cells have recently emerged as powerful tools for the discovery and investigation of recessive traits. Use of the haploid system provides unique genetic tractability and resolution. Upon positive selection, these screens typically employ analysis of loss-of-function (LOF) alleles and are thus limited to non-essential genes. Many relevant compounds, including anti-cancer therapeutics, however, target essential genes, precluding positive selection of LOF alleles. Here, we asked whether the use of random and saturating chemical mutagenesis might enable screens that identify essential biological targets of toxic compounds. We compare and contrast chemical mutagenesis with insertional mutagenesis. Selecting mutagenized cells with thapsigargin, an inhibitor of the essential Ca2+ pump SERCA2, insertional mutagenesis retrieved cell clones overexpressing SERCA2. With chemical mutagenesis, we identify six single amino acid substitutions in the known SERCA2-thapsigargin binding interface that confer drug resistance. In a second screen, we used the anti-cancer drug MG132/bortezomib (Velcade), which inhibits proteasome activity. Using chemical mutagenesis, we found 7 point mutations in the essential subunit Psmb5 that map to the bortezomib binding surface. Importantly, 4 of these had previously been identified in human tumors with acquired bortezomib resistance. Insertional mutagenesis did not identify Psmb5 in this screen, demonstrating the unique ability of chemical mutagenesis to identify relevant point mutations in essential genes. Thus, chemical mutagenesis in haploid embryonic stem cells can define the interaction of toxic small molecules with essential proteins at amino acid resolution, fully mapping small molecule-protein binding interfaces.

Oral Supplementation of Glucosamine Fails to Alleviate Acute Kidney Injury in Renal Ischemia-Reperfusion Damage.

Acute kidney injury is a leading contributor to morbidity and mortality in the ageing population. Proteotoxic stress response pathways have been suggested to contribute to the development of acute renal injury. Recent evidence suggests that increased synthesis of N-glycan precursors in the hexosamine pathway as well as feeding of animals with aminosugars produced in the hexosamine pathway may increase stress resistance through reducing proteotoxic stress and alleviate pathology in model organisms. As feeding of the hexosamine pathway metabolite glucosamine to aged mice increased their life expectancy we tested whether supplementation of this aminosugar may also protect mice from acute kidney injury after renal ischemia and reperfusion. Animals were fed for 4 weeks ad libitum with standard chow or standard chow supplemented with 0.5% N-acetylglucosamine. Preconditioning with caloric restriction for four weeks prior to surgery served as a positive control for protective dietary effects. Whereas caloric restriction demonstrated the known protective effect both on renal function as well as survival in the treated animals, glucosamine supplementation failed to promote any protection from ischemia-reperfusion injury. These data show that although hexosamine pathway metabolites have a proven role in enhancing protein quality control and survival in model organisms oral glucosamine supplementation at moderate doses that would be amenable to humans does not promote protection from ischemia-reperfusion injury of the kidney.

Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an α-glucosidase inhibitor or a Nrf2-inducer.

The National Institute on Aging Interventions Testing Program (ITP) evaluates agents hypothesized to increase healthy lifespan in genetically heterogeneous mice. Each compound is tested in parallel at three sites, and all results are published. We report the effects of lifelong treatment of mice with four agents not previously tested: Protandim, fish oil, ursodeoxycholic acid (UDCA) and metformin - the latter with and without rapamycin, and two drugs previously examined: 17-α-estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17-α-estradiol at a threefold higher dose robustly extended both median and maximal lifespan, but still only in males. The male-specific extension of median lifespan by NDGA was replicated at the original dose, and using doses threefold lower and higher. The effects of NDGA were dose dependent and male specific but without an effect on maximal lifespan. Protandim, a mixture of botanical extracts that activate Nrf2, extended median lifespan in males only. Metformin alone, at a dose of 0.1% in the diet, did not significantly extend lifespan. Metformin (0.1%) combined with rapamycin (14 ppm) robustly extended lifespan, suggestive of an added benefit, based on historical comparison with earlier studies of rapamycin given alone. The α-glucosidase inhibitor, acarbose, at a concentration previously tested (1000 ppm), significantly increased median longevity in males and 90th percentile lifespan in both sexes, even when treatment was started at 16 months. Neither fish oil nor UDCA extended lifespan. These results underscore the reproducibility of ITP longevity studies and illustrate the importance of identifying optimal doses in lifespan studies.

T-cadherin is critical for adiponectin-mediated cardioprotection in mice.

The circulating, adipocyte-secreted hormone adiponectin (APN) exerts protective effects on the heart under stress conditions. The receptors binding APN to cardiac tissue, however, have remained elusive. Here, we report that the glycosyl phosphatidylinositol­anchored cell surface glycoprotein T-cadherin (encoded by Cdh13) protects against cardiac stress through its association with APN in mice. We observed extensive colocalization of T-cadherin and APN on cardiomyocytes in vivo. In T-cadherin-deficient mice, APN failed to associate with cardiac tissue, and its levels dramatically increased in the circulation. Pressure overload stress resulted in exacerbated cardiac hypertrophy in T-cadherin-null mice and paralleled corresponding defects in mice lacking APN. During ischemia-reperfusion injury, the absence of T-cadherin increased infarct size similar to that in APN-null mice. Myocardial AMPK is a major downstream protective signaling target of APN. In both cardiac hypertrophy and ischemia-reperfusion models, T-cadherin was necessary for APN-dependent AMPK phosphorylation. In APN-null mice, recombinant adenovirus-expressed APN reduced exaggerated hypertrophy and infarct size and restored AMPK phosphorylation as previously reported. In contrast, rescue was ineffective in mice lacking T-cadherin in addition to APN. These data suggest that T-cadherin protects from stress-induced pathological cardiac remodeling by binding APN and activating its cardioprotective functions.

Adiponectin deficiency limits tumor vascularization in the MMTV-PyV-mT mouse model of mammary cancer.

PURPOSE: High levels of the fat-secreted cytokine adiponectin (APN) are present in the circulation of healthy people, whereas low levels correlate with an increased incidence of breast cancer in women. The current study experimentally probes the physiologic functions of APN in mammary cancer in a newly generated genetic mouse model. EXPERIMENTAL DESIGN: We established an APN null mouse model of mammary cancer by introducing the polyoma virus middle T (PyV-mT) oncogene expressed from mouse mammary tumor virus (MMTV) regulatory elements into APN null mice. MMTV-PyV-mT-induced tumors resemble ErbB2-amplified human breast cancers. We monitored tumor onset, kinetics, and animal survival, and analyzed vascular coverage, apoptosis, and hypoxia in sections from the primary tumors. Metastatic spreading was evaluated by analyses of the lungs. RESULTS: APN prominently localized to the vasculature in human and mouse mammary tumors. In APN null mice, MMTV-PyV-mT-induced tumors appeared with delayed onset and exhibited reduced growth rates. Affected animals survived control tumor-bearing mice by an average of 21 days. Pathologic analyses revealed reduced vascularization of APN null tumors along with increased hypoxia and apoptosis. At the experimental end point, APN null transgenic mice showed increased frequency of pulmonary metastases. CONCLUSION: The current work identifies a proangiogenic contribution of APN in mammary cancer that, in turn, affects tumor progression. APN interactions with vascular receptors may be useful targets for developing therapies aimed at controlling tumor vascularization in cancer patients.