Serine/Threonine Kinase 17A is a Novel Candidate for Therapeutic Targeting in Glioblastoma

STK17A is a relatively uncharacterized member of the death-associated protein family of serine/threonine kinases which have previously been associated with cell death and apoptosis. Our prior work established that STK17A is a novel p53 target gene that is induced by a variety of DNA damaging agents in a p53-dependent manner. In this study we have uncovered an additional, unanticipated role for STK17A as a candidate promoter of cell proliferation and survival in glioblastoma (GBM). Unexpectedly, it was found that STK17A is highly overexpressed in a grade-dependent manner in gliomas compared to normal brain and other cancer cell types with the highest level of expression in GBM. Knockdown of STK17A in GBM cells results in a dramatic alteration in cell shape that is associated with decreased proliferation, clonogenicity, migration, invasion and anchorage independent colony formation. STK17A knockdown also sensitizes GBM cells to genotoxic stress. STK17A overexpression is associated with a significant survival disadvantage among patients with glioma which is independent of age, molecular phenotype, IDH1 mutation, PTEN loss, and alterations in the p53 pathway and partially independent of grade. In summary, we demonstrate that STK17A provides a proliferative and survival advantage to GBM cells and is a potential target to be exploited therapeutically in patients with glioma

Activity of immunoproteasome inhibitor ONX-0914 in acute lymphoblastic leukemia expressing MLL–AF4 fusion protein

Proteasome inhibitors bortezomib and carfilzomib are approved for the treatment of multiple myeloma and mantle cell lymphoma and have demonstrated clinical efficacy for the treatment of acute lymphoblastic leukemia (ALL). The t(4;11)(q21;q23) chromosomal translocation that leads to the expression of MLL–AF4 fusion protein and confers a poor prognosis, is the major cause of infant ALL. This translocation sensitizes tumor cells to proteasome inhibitors, but toxicities of bortezomib and carfilzomib may limit their use in pediatric patients. Many of these toxicities are caused by on-target inhibition of proteasomes in non-lymphoid tissues (e.g., heart muscle, gut, testicles). We found that MLL–AF4 cells express high levels of lymphoid tissue-specific immunoproteasomes and are sensitive to pharmacologically relevant concentrations of specific immunoproteasome inhibitor ONX-0914, even in the presence of stromal cells. Inhibition of multiple active sites of the immunoproteasomes was required to achieve cytotoxicity against ALL. ONX-0914, an inhibitor of LMP7 (ß5i) and LMP2 (ß1i) sites of the immunoproteasome, and LU-102, inhibitor of proteasome ß2 sites, exhibited synergistic cytotoxicity. Treatment with ONX-0914 significantly delayed the growth of orthotopic ALL xenograft tumors in mice. T-cell ALL lines were also sensitive to pharmacologically relevant concentrations of ONX-0914. This study provides a strong rationale for testing clinical stage immunoproteasome inhibitors KZ-616 and M3258 in ALL

MiR-338-3p regulates neuronal maturation and suppresses glioblastoma proliferation

Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral sponge to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously in vivo. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation in vitro. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma

Seroprevalence of parvovirus B19 infection in daycare educators.

This study was undertaken to provide first-time estimates for the seroprevalence of parvovirus B19 infection among daycare educators in Montréal, Canada, and to identify factors associated with seropositivity. A cross-sectional design was used. Directors and educators from 81 daycare centres (DCCs) were surveyed about DCC and personal characteristics respectively, and serum samples from 477 female educators were tested for parvovirus B19 IgG antibodies. The seroprevalence of parvovirus B19 was 70%. Parvovirus B19 seropositivity was significantly associated with age and with working experience in DCCs, but the latter association was restricted to educators aged less than 40 years. In conclusion, working as a daycare educator appears to be associated with increased risk of acquiring parvovirus B19 infection, but this finding will require further investigation. Because of the large proportion of educators susceptible to acquiring parvovirus B19 infection, our findings also highlight the need for preventive measures

Serine/Threonine Kinase 17A Is a Novel Candidate for Therapeutic Targeting in Glioblastoma

<div><p>STK17A is a relatively uncharacterized member of the death-associated protein family of serine/threonine kinases which have previously been associated with cell death and apoptosis. Our prior work established that STK17A is a novel p53 target gene that is induced by a variety of DNA damaging agents in a p53-dependent manner. In this study we have uncovered an additional, unanticipated role for STK17A as a candidate promoter of cell proliferation and survival in glioblastoma (GBM). Unexpectedly, it was found that STK17A is highly overexpressed in a grade-dependent manner in gliomas compared to normal brain and other cancer cell types with the highest level of expression in GBM. Knockdown of STK17A in GBM cells results in a dramatic alteration in cell shape that is associated with decreased proliferation, clonogenicity, migration, invasion and anchorage independent colony formation. STK17A knockdown also sensitizes GBM cells to genotoxic stress. STK17A overexpression is associated with a significant survival disadvantage among patients with glioma which is independent of age, molecular phenotype, IDH1 mutation, PTEN loss, and alterations in the p53 pathway and partially independent of grade. In summary, we demonstrate that STK17A provides a proliferative and survival advantage to GBM cells and is a potential target to be exploited therapeutically in patients with glioma. </p> </div

STK17A is overexpressed in GBM.

<p><b>A</b>, Data from Sun Brain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081803#B19" target="_blank">19</a>], and TCGA Brain (<a href="https://tcga-data.nci.nih.gov/tcga/tcgahome2.jsp" target="_blank"><u>https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp</u></a>) comparing microarray-based STK17A expression from clinical nonmalignant brain specimens (Norm. Brain) and clinical GBM indicating high expression of STK17A in GBM. <b>B</b>, Real-time PCR analysis of STK17A expression in GBM cell lines compared to expression in other cancer types and normal brain. Expression is normalized to GAPDH. Bars are the average of triplicate or duplicate biological replicates except for normal brain RNA which is the average of technical duplicates. Error bars are SD. All cell lines are human. Non-GBM cell lines are as follows: NT2 (NT2/D1), embryonal carcinoma; U2OS, osteosarcoma; H23, A549, and Hop62, lung adenocarcinomas; MCF7, breast cancer. <b>C</b>, Real-time PCR analysis of STK17A expression in normal brain mRNA and four clinical GBM samples from Dartmouth Hitchcock Medical Center. Expression is normalized to GAPDH. The bars represent the averages of technical duplicate determinations. </p

STK17A expression in glioma is associated with high grade and decreased survival.

<p><b>A</b>, Expression data was downloaded from the Rembrandt and TCGA databases and data from Sun Brain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081803#B19" target="_blank">19</a>] was downloaded from Oncomine and grouped according to grade. Rembrandt has 21 normal, 99 grade II, 85 grade III and 130 grade IV samples. TCGA has 10 normal, 7 grade II, 20 grade III and 482 grade IV samples and Sun Brain has 26 normal, 45 grade II, 31 grade III and 81 grade IV samples. Error bars are SEM. *, p < 0.02. <b>B</b>, STK17A expression is associated with poor overall survival in gliomas. Kaplan-Meier log-rank tests were performed on data obtained from the TCGA database. In all cases high and low expressing groups were divided at the median. All glioma and low grade glioma expression was from RNA-seq data while GBM expression was from Affymetrix microarray data. </p

STK17A knockdown decreases oncogenic properties of GBM cells.

<p><b>A</b>, STK17A knockdown decreases soft agar colony formation. U87 control or U87 STK17A knockdown cells were suspended in soft agar and cells were stained with MTT reagent after 2 weeks of culture. Representative of two independent experiments. <b>B</b>, STK17A knockdown decreases clonogenicity of GBM cells. U87 control or STK17A U87 knockdown cells were plated and stained with Giemsa after 10 days of cell culture. Representative of three independent experiments. <b>C</b>, STK17A knockdown sensitizes GBM cells to cisplatin and temozolomide. Left, Dose response after 3 days of cisplatin treatment of U87 or U251 control versus STK17A knockdown cells. Cell proliferation and survival was measured with Cell-Titer Glo reagent. Data points are the average of biological triplicates and error bars are SD. *, p < 0.05. Representative of three independent experiments. Right, Dose response after 3 days of temozolomide treatment of U87 control versus U87 STK17A knockdown cells. Cell proliferation and survival was measured with Cell-Titer Glo reagent. Data points are the average of biological triplicates and error bars are SD. *, p< 0.05. Representative of two independent experiments. </p

STK17A knockdown results in the formation of actin stress fibers and inhibition of cell motility and invasion.

<p><b>A</b>, Representative fluorescent images of U87 cells stably expressing control shRNA and two distinct STK17A shRNAs. Actin is stained green and nuclei are stained blue. Note the larger size of STK17A knockdown cells and the presence of actin stress fibers. Extent of knockdown is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081803#pone-0081803-g003" target="_blank">Figure 3</a>. This morphologic phenotype was apparent in independent derivations of the cell lines. Pictures were taken at 20X magnification on a NIKON ELWD fluorescent microscope. <b>B</b>, STK17A knockdown decreases cell migration and invasion of U87 cells. Cell migration and invasion was assessed as described in Methods. Bars are the average of biological triplicates and error bars are SD. *, p < 0.05. Representative of two independent experiments.</p