Cardiac Glycosides Induce Cell Death in Human Cells by Inhibiting General Protein Synthesis

BACKGROUND: Cardiac glycosides are Na(+)/K(+)-pump inhibitors widely used to treat heart failure. They are also highly cytotoxic, and studies have suggested specific anti-tumor activity leading to current clinical trials in cancer patients. However, a definitive demonstration of this putative anti-cancer activity and the underlying molecular mechanism has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: Using an unbiased transcriptomics approach, we found that cardiac glycosides inhibit general protein synthesis. Protein synthesis inhibition and cytotoxicity were not specific for cancer cells as they were observed in both primary and cancer cell lines. These effects were dependent on the Na(+)/K(+)-pump as they were rescued by expression of a cardiac glycoside-resistant Na(+)/K(+)-pump. Unlike human cells, rodent cells are largely resistant to cardiac glycosides in vitro and mice were found to tolerate extremely high levels. CONCLUSIONS/SIGNIFICANCE: The physiological difference between human and mouse explains the previously observed sensitivity of human cancer cells in mouse xenograft experiments. Thus, published mouse xenograft models used to support anti-tumor activity for these drugs require reevaluation. Our finding that cardiac glycosides inhibit protein synthesis provides a mechanism for the cytotoxicity of CGs and raises concerns about ongoing clinical trials to test CGs as anti-cancer agents in humans

Normal and pathological erythropoiesis in adults: from gene regulation to targeted treatment concepts

Pathological erythropoiesis with consequent anemia is a leading cause of symptomatic morbidity in internal medicine. The etiologies of anemia are complex and include reactive as well as neoplastic conditions. Clonal expansion of erythroid cells in the bone marrow may result in peripheral erythrocytosis and polycythemia but can also result in anemia when clonal cells are dysplastic and have a maturation arrest that leads to apoptosis and hinders migration, a constellation typically seen in the myelodysplastic syndromes. Rarely, clonal expansion of immature erythroid blasts results in a clinical picture resembling erythroid leukemia. Although several mechanisms underlying normal and abnormal erythropoiesis and the pathogenesis of related disorders have been deciphered in recent years, little is known about specific markers and targets through which prognosis and therapy could be improved in anemic or polycythemic patients. In order to discuss new markers, targets and novel therapeutic approaches in erythroid disorders and the related pathologies, a workshop was organized in Vienna in April 2017. The outcomes of this workshop are summarized in this review, which includes a discussion of new diagnostic and prognostic markers, the updated WHO classification, and an overview of new drugs used to stimulate or to interfere with erythropoiesis in various neoplastic and reactive conditions. The use and usefulness of established and novel erythropoiesis-stimulating agents for various indications, including myelodysplastic syndromes and other neoplasms, are also discussed

Ubiquitin-Specific Protease 4 Inhibits Mono-Ubiquitination of the Master Growth Factor Signaling Kinase PDK1

BACKGROUND: Phosphorylation by the phospho-inositide-dependent kinase 1 (PDK1) is essential for many growth factor-activated kinases and thus plays a critical role in various processes such as cell proliferation and metabolism. However, the mechanisms that control PDK1 have not been fully explored and this is of great importance as interfering with PDK1 signaling may be useful to treat diseases, including cancer and diabetes. METHODOLOGY/PRINCIPAL FINDINGS: In human cells, few mono-ubiquitinated proteins have been described but in all cases this post-translational modification has a key regulatory function. Unexpectedly, we find that PDK1 is mono-ubiquitinated in a variety of human cell lines, indicating that PDK1 ubiquitination is a common and regulated process. Ubiquitination occurs in the kinase domain of PDK1 yet is independent of its kinase activity. By screening a library of ubiquitin proteases, we further identify the Ubiquitin-Specific Protease 4 (USP4) as an enzyme that removes ubiquitin from PDK1 in vivo and in vitro and co-localizes with PDK1 at the plasma membrane when the two proteins are overexpressed, indicating direct deubiquitination. CONCLUSIONS: The regulated mono-ubiquitination of PDK1 provides an unanticipated layer of complexity in this central signaling network and offers potential novel avenues for drug discovery

CDK6 Inhibition: A Novel Approach in AML Management

Acute myeloid leukemia (AML) is a complex disease with an aggressive clinical course and high mortality rate. The standard of care for patients has only changed minimally over the past 40 years. However, potentially useful agents have moved from bench to bedside with the potential to revolutionize therapeutic strategies. As such, cell-cycle inhibitors have been discussed as alternative treatment options for AML. In this review, we focus on cyclin-dependent kinase 6 (CDK6) emerging as a key molecule with distinct functions in different subsets of AML. CDK6 exerts its effects in a kinase-dependent and -independent manner which is of clinical significance as current inhibitors only target the enzymatic activity

Targeted Protein Degradation: Clinical Advances in the Field of Oncology

The field of targeted protein degradation (TPD) is a rapidly developing therapeutic modality with the promise to tame disease-relevant proteins in ways that are difficult or impossible to tackle with other strategies. While we move into the third decade of TPD, multiple degrader drugs have entered the stage of the clinic and many more are expected to follow. In this review, we provide an update on the most recent advances in the field of targeted degradation with insights into possible clinical implications for cancer prevention and treatment

Therapeutic Vulnerabilities in <i>FLT3</i>-Mutant AML Unmasked by Palbociclib

While significant progress has been made in the treatment of acute myeloid leukemia (AML), not all patients can be cured. Mutated in about 1/3 of de novo AML, the FLT3 receptor tyrosine kinase is an attractive target for drug development, activating mutations of the FLT3 map to the juxtamembrane domain (internal tandem duplications, ITD) or the tyrosine kinase domain (TKD), most frequently at codon D835. While small molecule tyrosine kinase inhibitors (TKI) effectively target ITD mutant forms, those on the TKD are not responsive. Moreover, FLT3 inhibition fails to induce a persistent response in patients due to mutational resistance. More potent compounds with broader inhibitory effects on multiple FLT3 mutations are highly desirable. We describe a critical role of CDK6 in the survival of FLT3+ AML cells as palbociclib induced apoptosis not only in FLT3&#8315;ITD+ cells but also in FLT3&#8315;D835Y+ cells. Antineoplastic effects were also seen in primary patient-derived cells and in a xenograft model, where therapy effectively suppressed tumor formation in vivo at clinically relevant concentrations. In cells with FLT3&#8315;ITD or -TKD mutations, the CDK6 protein not only affects cell cycle progression but also transcriptionally regulates oncogenic kinases mediating intrinsic drug resistance, including AURORA and AKT&#8212;a feature not shared by its homolog CDK4. While AKT and AURORA kinase inhibitors have significant therapeutic potential in AML, single agent activity has not been proven overly effective. We describe synergistic combination effects when applying these drugs together with palbociclib which could be readily translated to patients with AML bearing FLT3&#8315;ITD or &#8315;TKD mutations. Targeting synergistically acting vulnerabilities, with CDK6 being the common denominator, may represent a promising strategy to improve AML patient responses and to reduce the incidence of selection of resistance-inducing mutations

Ub-PDK1 localizes to all cellular compartments, binds phospholipids and mono-ubiquitination is not dependent on kinase activity.

<p><b>A</b>: HEK293T cells were co-transfected with wild type V5-PDK1 (WT) or the indicated lysine mutants and GFP. Whole cell extracts were blotted and probed with anti-V5 and GFP that served as a transfection efficiency control. <b>B</b>: Cell fractionation analysis of HEK293T cells transfected with V5-tagged PDK1 (Cyt = cytoplasmic, Mem = membrane bound, Nuc = nuclear). Ub-PDK1 was detected with a V5 antibody. The HSP90, Histone H1 (H1) and Actin antibodies were used as controls. <b>C</b>: HEK293T cells were transfected as indicated and PDK1 was pulled down using phospholipid-coated (PIP3) or control beads. Pull-downs and input were probed with anti-V5 antibody. <b>D</b>: HEK293T cells were transfected and the catalytically inactive mutant K110R of PDK1 was immunoprecipitated with anti-V5 beads and probed with anti-V5 antibody. The wild type construct (WT, V5-PDK1) was used as a control. <b>E</b>: HEK293T cells were transfected as indicated and PDK1 was immunoprecipitated with anti-V5 beads. Immunoprecipitations (IP) were analyzed with an antibody recognizing phosphorylated PDK1 at the Ser241 site.</p

USP4 and PDK1 interact and co-localize at the plasma membrane.

<p><b>A</b>: HEK293T cells were transfected as indicated and PDK1 was immunoprecipitated with anti-V5 beads. Immunoprecipitations (IP) and input were probed with anti-V5 and MYC antibodies. <b>B</b>: Confocal images from transfected U2OS cells stained with V5-PDK1 (green) and MYC-USP4 (red) antibodies. DNA was visualized by DAPI.</p

The kinase domain of PDK1 is mono-ubiquitinated.

<p><b>A</b>: Overview of the PDK1 constructs used in the figure. PDK1 consists of an amino-terminal kinase domain (KD) and a carboxyl-terminal Pleckstrin Homology (PH) domain. The K-less mutant has all 27 lysine residues (K) mutated to arginines (R). <b>B</b>: HEK293T cells were transfected as indicated and PDK1 was immunoprecipitated with anti-V5 beads. Immunoprecipitations (IP) and input were immunoblotted with anti-V5 antibody. <b>C</b>: HEK293T cells were transfected as indicated and proteins were immunoprecipitated with anti-V5 beads. Immunoprecipitations (IP) were immunoblotted with V5 and HA antibodies. <b>D</b>: HEK293T cells were transfected and the lysine-less mutant of PDK1 was immunoprecipitated with anti-V5 beads and probed with anti-V5 antibody. The wild type construct (WT, V5-PDK1) was used as a control.</p