Identification of novel small molecules that inhibit STAT3-dependent transcription and function

Activation of Signal Transducer and Activator of Transcription 3 (STAT3) has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z’ = 0,8). This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set). Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents

Design and evaluation of drug delivery vehicles

A crucial aspect of drug delivery is efficient transport to the site of action. Thus, there is a need to design and evaluate new delivery vehicles. In this thesis two delivery vehicles, cell-penetrating peptides and bacterial ghosts, were evaluated. The understanding of the internalization and degradation kinetics of cell-penetrating peptides is important for the practical aspects of cargo delivery since peptides have a notorious reputation of being rapidly degraded. If the cell-penetrating peptide remains intact inside the cellular environment, there is a possibility that the peptide-cargo conjugate leaks back to the extracellular environment. However, if it is degraded outside the cell, the cargo will never be delivered. In order to improve uptake efficiency and to be able to foresee side effects, the translocation mechanism needs to be fully elucidated. Data gathered from the first two papers led to the proposal of a new me-chanism involved in cell-penetrating peptide uptake: the membrane repair response, a resealing mechanism rapidly patching up broken membranes. This mechanism could explain the divergence in perception concerning the uptake pathways. Furthermore a new assay to produce the second delivery vehicle, bacterial ghosts, was developed based on data from the cell-penetrating peptide investigations. Bacterial ghosts are dead bacteria devoid of cytoplasmic contents but still retaining their structural and morphological characteristics, after protein E lysis of the bacterial cell membrane. By using a cell-penetrating peptide with antimicrobial effects, a new rapid peptide-based strategy to produce ghosts was developed and the capability to deliver plasmid DNA into the cell for expression was evaluated

PLK1 as a cooperating partner for BCL2-mediated antiapoptotic program in leukemia

Abstract The deregulation of BCL2 family proteins plays a crucial role in leukemia development. Therefore, pharmacological inhibition of this family of proteins is becoming a prevalent treatment method. However, due to the emergence of primary and acquired resistance, efficacy is compromised in clinical or preclinical settings. We developed a drug sensitivity prediction model utilizing a deep tabular learning algorithm for the assessment of venetoclax sensitivity in T-cell acute lymphoblastic leukemia (T-ALL) patient samples. Through analysis of predicted venetoclax-sensitive and resistant samples, PLK1 was identified as a cooperating partner for the BCL2-mediated antiapoptotic program. This finding was substantiated by additional data obtained through phosphoproteomics and high-throughput kinase screening. Concurrent treatment using venetoclax with PLK1-specific inhibitors and PLK1 knockdown demonstrated a greater therapeutic effect on T-ALL cell lines, patient-derived xenografts, and engrafted mice compared with using each treatment separately. Mechanistically, the attenuation of PLK1 enhanced BCL2 inhibitor sensitivity through upregulation of BCL2L13 and PMAIP1 expression. Collectively, these findings underscore the dependency of T-ALL on PLK1 and postulate a plausible regulatory mechanism

Primers used for qRT-PCR.

<p>Primers used for qRT-PCR.</p

Characterization of the four hit compounds.

<p><b>(A-D)</b> Compounds KI1, KI4, KI12 and KI16 and their corresponding activities in the luciferase reporter assays (center) and cell viability assays (right). Luciferase assays were performed in A4wt cells stimulated with IL6 (grey) and in A4 cells stimulated with IFNγ (red). Viability assays were conducted over 48 hours in A4wt cells (grey) and A4 cells (red). <b>(E)</b> Luciferase IC₅₀ values for lead compounds. Curves were fit using GraphPad and error bars indicate the 95% confidence intervals. <b>(F)</b> Cell viability EC₅₀ values in A4 and A4wt cells using CellTiterGlo assay (Promega Biotech AB, Sweden). Curves were fit using GraphPad and error bars indicate the 95% confidence intervals.</p

The effect of the lead compounds on the IL6-JAK-STAT pathway.

<p><b>(A)</b> A4wt or A4 cell lines were pre-treated with the indicated compounds (5 and 10μM) for 30 min followed by stimulation with IL6+sIL6R (50 and 100 ng/mL, respectively) for 30 min. Levels of phosphorylated STAT3 (pTyr705) and STAT1 (pTyr701) and total levels of these proteins were determined by Western blotting analysis. GAPDH was used as a loading control. Both left and right panels of western blots were performed simultaneously. <b>(B)</b> A4wt and A4 cell lines were pre-treated with the indicated compounds (5 and 10μM) for 30 min followed by stimulation with IFNγ (40 IU/mL) for 30 min. Levels of phospho-STAT1 and total STAT1 were determined by Western blotting analysis. GAPDH was used as a loading control. <b>(C)</b> A4wt cells were pre-treated with the compounds KI1, KI4, KI12 and KI16 in the concentration of 10 μM and then treated with IL6 + sIL6R (50 ng/mL and 100 ng/mL respectively) for 30 min. The cell pellets were lysed and pJAK1, pJAK2 and total JAK1 and JAK2 levels were assessed by Western blotting. GAPDH was used as a loading control. <b>(D)</b> A4wt cells were stimulated with either IL6+sIL6R (50 and 100 ng/mL, respectively) or IFNγ (40 IU/mL) for 4 h. Expression of indicated STAT3 target genes was assessed by qRT-PCR. The data represents mean of duplicates ± SD. <b>(E)</b> A4wt cells were pretreated with the indicated compounds (5 and 10 μM) for 30 min followed by IL6+sIL6R stimulation for 4 h (as in <b>D</b>). The expression of indicated STAT3 target genes was determined by qRT-PCR. The data is normalized to β-actin expression and presented as fold expression relative to the untreated control. The data represents mean of duplicates ± SD.</p

Differential effect of the lead compounds on viability of STAT3-dependent and STAT3-independent cancer cell lines.

<p><b>(A)</b> Cell pellets from the prostate cancer cells lines PC3 and DU145, and the breast cancer cell lines MCF7 and MDA-MB468, were lysed and subjected to Western blotting analysis. Antibodies against pSerSTAT3, pTyrSTAT3, pTyrSTAT1 and the corresponding antibodies for the total levels of these proteins were used. GAPDH was used as a loading control. <b>(B)</b> PC3, DU145, MCF7 and MDA-MB468 cells were seeded in 384-well white plates using Multidrop. Cells were treated with the range of compounds concentration using Echo liquid dispenser. Cell viability was assessed after 48h using CellTiterGlo viability assay (Promega) according to the instructions of the manufacturer. The viability of the DMSO-treated cells was set as 100%. The data represents IC50 values for the four cell lines calculated from three replicates ± SD. <b>(C, D)</b> DU145 <b>(C)</b> and MDA-MB468 <b>(D)</b> cells were treated with the indicated compounds for 4 h. Levels of phospho-STAT3 and total STAT3 were determined by Western blotting analysis. GAPDH was used as a loading control. <b>(E)</b> DU145 cell line was transfected with 50 nM scrambled siRNA or STAT3 siRNA for 48 h or treated with the indicated compounds for 16 h and 24h, respectively. The concentrations of the compounds were taken from the IC₅₀ values for viability (<b>3B</b>). Expression of the indicated STAT3 target genes was analysed by qRT-PCR. The values are normalized to the expression of GUSB and presented as a relative expression to untreated controls. Error bars represent mean + SE from three independent experiments. <b>(F</b>) A wound healing assay was performed using DU145 cells treated with the indicated compounds at 20 μM. Migration into the denuded area was monitored after 4, 8 and 24 h. The data represents mean of triplicates ± SE from three independent experiments. * p < 0.05.</p