Genome-wide screens identify specific drivers of mutant hTERT promoters

Cancer-specific hTERT promoter mutations reported in 19% of cancers result in enhanced telomerase activity. Understanding the distinctions between transcriptional regulation of wild-type (WT) and mutant (Mut) hTERT promoters may open up avenues for development of inhibitors which specially block hTERT expression in cancer cells. To comprehensively identify physiological regulators of WT- or Mut-hTERT promoters, we generated several isogenic reporter cells driven by endogenous hTERT loci. Genome-wide CRISPR-Cas9 and small interfering RNA screens using these isogenic reporter lines identified specific regulators of Mut-hTERT promoters. We validate and characterize one of these hits, namely, MED12, a kinase subunit of mediator complex. We demonstrate that MED12 specifically drives expression of hTERT from the Mut-hTERT promoter by mediating long-range chromatin interaction between the proximal Mut-hTERT promoter and T-INT1 distal regulatory region 260 kb upstream. Several hits identified in our screens could serve as potential therapeutic targets, inhibition of which may specifically block Mut-hTERT promoter driven telomerase reactivation in cancers.

A biodegradable amphiphilic poly(aminoester) dendrimer for safe and effective siRNA delivery

Small interfering RNA (siRNA)-based therapeutics represent a novel and compelling drug modality, provided that safe and competent vectors are available for their delivery. Here, we report a biodegradable amphiphilic poly(aminoester)...

Sulfonated Amphiphilic Poly(α)glutamate Amine—A Potential siRNA Nanocarrier for the Treatment of Both Chemo-Sensitive and Chemo-Resistant Glioblastoma Tumors

Development of chemo-resistance is a major challenge in glioblastoma (GB) treatment. This phenomenon is often driven by increased activation of genes associated with DNA repair, such as the alkyl-removing enzyme O6-methylguanine-DNA methyltransferase (MGMT) in combination with overexpression of canonical genes related to cell proliferation and tumor progression, such as Polo-like kinase 1 (Plk1). Hereby, we attempt to sensitize resistant GB cells using our established amphiphilic poly(α)glutamate (APA): small interfering RNA (siRNA) polyplexes, targeting Plk1. Furthermore, we improved brain-targeting by decorating our nanocarrier with sulfonate groups. Our sulfonated nanocarrier showed superior selectivity towards P-selectin (SELP), a transmembrane glycoprotein overexpressed in GB and angiogenic brain endothelial cells. Self-assembled polyplexes of sulfonated APA and siPlk1 internalized into GB cells and into our unique 3-dimensional (3D) GB spheroids inducing specific gene silencing. Moreover, our RNAi nanotherapy efficiently reduced the cell viability of both chemo-sensitive and chemo-resistant GB cells. Our developed sulfonated amphiphilic poly(α)glutamate nanocarrier has the potential to target siRNA to GB brain tumors. Our findings may strengthen the therapeutic applications of siRNA for chemo-resistant GB tumors, or as a combination therapy for chemo-sensitive GB tumors.

GPER mediated estrogenic amelioration of sodium channel dysfunction in stressed human induced pluripotent stem cell-derived cardiomyocytes

Stress-induced excessive activation of the adrenergic system or changes in estrogen levels promote the occurrence of arrhythmias. Sodium channel, a responder to β-adrenergic stimulation, is involved in stress-induced cardiac electrophysiological abnormalities. However, it has not been established whether estrogen regulates sodium channels during acute stress. Our study aimed to explore whether voltage-gated sodium channels play roles in the rapid regulation of various concentrations of estrogen in stressed human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and reveal the possible mechanism of estrogen signaling pathway modulating stress. An isoproterenol-induced stress model of hiPSC-CMs was pre-incubated with β-Estradiol at different concentrations (0.01 nmol/L, 1 nmol/L, and 100 nmol/L). Action potential (AP) and sodium currents were detected by patch clamp. The G protein-coupled estrogen receptor (GPER)-specific effect was determined with agonists G1, antagonists G15 and small interfering RNA. β-Estradiol at concentrations of 0.01 nmol/L, 1 nmol/L, and 100 nmol/L increased the peak sodium current and prolonged AP duration (APD) at 1 nmol/L. Stress increased peak sodium current, late sodium current, and shortened APD. The effects of stress on sodium currents and APD were eliminated by β-Estradiol. Activation of GPER by G1 exhibited similar effects as β-Estradiol, while inhibition of GPER with G15 and small interfering RNA ameliorated estrogenic actions. Estrogen, antagonized the stress-related abnormal electrical activity, and through GPER alleviated sodium channel dysfunctions in stress state in hiPSC-CMs. These results provide a novel mechanism through which estrogenic rapid signaling against stress by regulating ion channels.