Dual TTK/PLK1 inhibition has potent anticancer activity in TNBC as monotherapy and in combination.

Background: Threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1) are common essential kinases that collaborate in activating the spindle assembly checkpoint (SAC) at the kinetochore, ensuring appropriate chromosome alignment and segregation prior to mitotic exit. Targeting of either TTK or PLK1 has been clinically evaluated in cancer patients; however, dual inhibitors have not yet been pursued. Here we present the in vitro and in vivo characterization of a first in class, dual TTK/PLK1 inhibitor (BAL0891). Methods: Mechanism of action studies utilized biochemical kinase and proteomics-based target-engagement assays. Cellular end-point assays included immunoblot- and flow cytometry-based cell cycle analyses and SAC integrity evaluation using immunoprecipitation and immunofluorescence approaches. Anticancer activity was assessed in vitro using cell growth assays and efficacy was evaluated, alone and in combination with paclitaxel and carboplatin, using mouse models of triple negative breast cancer (TNBC). Results: BAL0891 elicits a prolonged effect on TTK, with a transient activity on PLK1. This unique profile potentiates SAC disruption, forcing tumor cells to aberrantly exit mitosis with faster kinetics than observed with a TTK-specific inhibitor. Broad anti-proliferative activity was demonstrated across solid tumor cell lines in vitro. Moreover, intermittent intravenous single-agent BAL0891 treatment of the MDA-MB-231 mouse model of TNBC induced profound tumor regressions associated with prolonged TTK and transient PLK1 in-tumor target occupancy. Furthermore, differential tumor responses across a panel of thirteen TNBC patient-derived xenograft models indicated profound anticancer activity in a subset (~40%). Using a flexible dosing approach, pathologically confirmed cures were observed in combination with paclitaxel, whereas synergy with carboplatin was schedule dependent. Conclusions: Dual TTK/PLK1 inhibition represents a novel approach for the treatment of human cancer, including TNBC patients, with a potential for potent anticancer activity and a favorable therapeutic index. Moreover, combination approaches may provide an avenue to expand responsive patient populations.

Bioinformatics prediction of overlapping frameshifted translation products in mammalian transcripts.

BACKGROUND: Exceptionally, a single nucleotide sequence can be translated in vivo in two different frames to yield distinct proteins. In the case of the G-protein alpha subunit XL-alpha-s transcript, a frameshifted open reading frame (ORF) in exon 1 is translated to yield a structurally distinct protein called Alex, which plays a role in platelet aggregation and neurological processes. We carried out a novel bioinformatics screen for other possible dual-frame translated sequences, based on comparative genomics. RESULTS: Our method searched human, mouse and rat transcripts in frames +1 and -1 for ORFs which are unusually well conserved at the amino acid level. We name these conserved frameshifted overlapping ORFs 'matreshkas' to reflect their nested character. Select findings of our analysis revealed that the G-protein coupled receptor GPR27 is entirely contained within a frame -1 matreshka, thrombopoietin contains a matreshka which spans ~70% of its length, platelet glycoprotein IIIa (ITGB3) contains a matreshka with the predicted characteristics of a secreted peptide hormone, while the potassium channel KCNK12 contains a matreshka spanning >400 amino acids. CONCLUSION: Although the in vivo existence of translated matreshkas has not been experimentally verified, this genome-wide analysis provides strong evidence that substantial overlapping coding sequences exist in a number of human and rodent transcripts.

A microarray-based, integrated approach to identify novel regulators of cancer drug response and apoptosis.

DNA microarrays are powerful tools for the analysis of gene expression on a genomic scale. The importance of individual regulatory events for the process under study can however not be deduced unequivocally without additional experiments. We devised a strategy to identify central regulators of cancer drug responses by combining the results of microarray experiments with efficient methods for phenotypic testing of candidate genes. We exposed murine FL5.12 pro-B cells to cisplatin, camptothecin, methotrexate or paclitaxel, respectively and analysed the patterns of gene expression with cDNA microarrays. Drug-specific regulatory events as well as intersections between different apoptotic pathways, including previously studied responses to staurosporine and interleukin-3 (IL-3) deprivation, were identified. Genes shared by at least three pathways were chosen for further analysis. Ectopic expression of three such genes, TEAP, GP49B, and Lipin1 was found to have an anti-proliferative effect on pro-B cells. Interestingly, we identified hemoglobin alpha as a strong pro-apoptotic regulator. While hemoglobin-expressing cells were growing normally in the presence of IL-3, they displayed accelerated apoptosis with similar kinetics as Bax overexpressing cells upon IL-3 removal. The pro-apoptotic effect of hemoglobin was suppressed by Bcl-2 and was characterized by enhanced stimulation of caspase activity.