Kinome-level screening identifies inhibition of polo-like kinase-1 (PLK1) as a target for enhancing non-viral transgene expression

Abstract

Human cells contain hundreds of kinase enzymes that regulate several cellular processes, which likely include transgene delivery and expression. We identified several kinases that influence gene delivery and/or expression by performing a kinome-level screen in which, we identified small-molecule kinase inhibitors that significantly enhanced non-viral (polymer-mediated) transgene (luciferase) expression in cancer cells. The strongest enhancement was observed with several small-molecule inhibitors of Polo-like Kinase 1 (PLK 1) (e.g., HMN-214 and BI 2536), which enhanced luciferase expression up to 30-fold by arresting cells in the G2/M phase of the cell cycle and influencing intracellular trafficking of plasmid DNA. Knockdown of PLK 1 using an shRNA-expressing lentivirus further confirmed the enhancement of polymer-mediated transgene expression. In addition, pairwise and three-way combinations of PLK1 inhibitors with the histone deacetylase-1 (HDAC-1) inhibitor Entinostat and the JAK/STAT inhibitor AG-490 enhanced luciferase expression to levels significantly higher than individual drug treatments acting alone. These findings indicate that inhibition of specific intracellular kinases (e.g., PLK1) can significantly enhance non-viral transgene expression for applications in biotechnology and medicine.

Small Molecule Inhibition of the Innate Immune Response Increases Transgene Expression

“…Interestingly, it has also been shown that inhibition of JAK/STAT signaling with the JAK inhibitor AG-490 significantly increases luciferase expression in PC3 cells. 42 Altogether, these results show that plasmid DNA activates an innate immune response that increases IL-6 expression and hinders transgene expression, but inhibition of different parts of this pathway can restore transgene expression.…”

Nonviral gene delivery to T cells with Lipofectamine LTX

“…In addition to vehicles and promoters, the effects of several other variables were also tested to optimize transfection efficiency in Jurkat T cells. For example, cells were treated with small molecule inhibitors that have previously been shown to enhance transgene expression in other cell types (e.g., BX795, MS‐275, AG‐490, and HMN‐214) (Christensen et al, 2015; Sutlu et al, 2012). One of the inhibitors (iCRT14) enhanced luciferase expression approximately threefold, but did not significantly enhance transfection efficiency (i.e., %EGFP + cells, shown in Figure S2).…”

Alveolar rhabdomyosarcoma: morphoproteomics and personalized tumor graft testing further define the biology of PAX3-FKHR(FOXO1) subtype and provide targeted therapeutic options

“…In this context, the following agents should act to address the biology of ARMS by acting at genomic and proteomic levels: 1) Entinostat by virtue of its ability to: a) inhibit class I HDAC [32] and thereby, to interrupt the YY1-EZH2-HDAC1 inhibition of miR-29b2 and mir-29c allowing arrest of ARMS cell proliferation and promoting differentiation [11–17, 28]; and b) its ability to effect direct transcriptional supression of PAX3:FOXO1 [33] and to inhibit the phosphorylative activation of PAX3—FOXO1 by polo-like kinase (PLK)1 [29, 34, 35]; 2) celecoxib inhibits the NF-kappaB pathway at multiple points [36] and should reduce the overproduction of YY1; 3) sulforaphane, a nutraceutical suppresses polycomb group protein level including EZH2 [37] and this should promote alveolar rhabdomyosarcoma's differentiation to a more benign form, induce apoptosis in the tumor cells [38] and reduce the survival of alveolar rhabdomyosarcoma leading to elimination of the tumor (in the latter study, sulforaphane was also shown to decrease the mRNA and protein levels of PAX3-FKHR, MYCN and MET in ARMS cells); 4) retinoic acid upregulates miR-214 which downregulates EZH2 protein in embryonic stem cells and miR-214-mediated EZH2 protein reduction accelerates skeletal muscle cell differentiation [39] (Both all-trans retinoic acid, ATRA and 9-cis retinoic acid suppressed the cell growth of alveolar rhabdomyosarcoma with evidence of a differentiating effect [40]. ATRA increased the expression of some genes associated with muscle differentiation and slowed the proliferation and promoted a more differentiated myogenic phenotype in ARMS cell lines [41].…”