Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy

BACKGROUND: The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. One of the best characterised checkpoint pathways is the spindle assembly checkpoint, which prevents anaphase onset until the appropriate attachment and tension across kinetochores is achieved. MPS1 kinase activity is essential for the activation of the spindle assembly checkpoint and has been shown to be deregulated in human tumours with chromosomal instability and aneuploidy. Therefore, MPS1 inhibition represents an attractive strategy to target cancers. METHODS: To evaluate CCT271850 cellular potency, two specific antibodies that recognise the activation sites of MPS1 were used and its antiproliferative activity was determined in 91 human cancer cell lines. DLD1 cells with induced GFP-MPS1 and HCT116 cells were used in in vivo studies to directly measure MPS1 inhibition and efficacy of CCT271850 treatment. RESULTS: CCT271850 selectively and potently inhibits MPS1 kinase activity in biochemical and cellular assays and in in vivo models. Mechanistically, tumour cells treated with CCT271850 acquire aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment because of abrogation of the mitotic checkpoint, leading to cell death. We demonstrated a moderate level of efficacy of CCT271850 as a single agent in a human colorectal carcinoma xenograft model. CONCLUSIONS: CCT271850 is a potent, selective and orally bioavailable MPS1 kinase inhibitor. On the basis of in vivo pharmacodynamic vs efficacy relationships, we predict that more than 80% inhibition of MPS1 activity for at least 24 h is required to achieve tumour stasis or regression by CCT271850

Rapid Discovery of Pyrido[3,4- d ]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach

Monopolar spindle 1 (MPS1) plays a central role in the transition of cells from metaphase to anaphase and is one of the main components of the spindle assembly checkpoint. Chromosomally unstable cancer cells rely heavily on MPS1 to cope with the stress arising from abnormal numbers of chromosomes and centrosomes and are thus more sensitive to MPS1 inhibition than normal cells. We report the discovery and optimization of a series of new pyrido[3,4-d]pyrimidine based inhibitors via a structure-based hybridization approach from our previously reported inhibitor CCT251455 and a modestly potent screening hit. Compounds in this novel series display excellent potency and selectivity for MPS1, which translates into biomarker modulation in an in vivo human tumor xenograft mode

Aurora B expression directly correlates with prostate cancer malignancy and influence prostate cell proliferation.

BACKGROUND: Chromosomal instability is one of the most common features of prostate cancer (PC), especially in advanced stages. Recent studies suggest that defects in mitotic checkpoints play a role in carcinogenesis. Lack of mitotic regulation induces aneuploidy in cancer cells acting thereafter as a driving force for malignant progression. Serine/threonine protein kinases of the Aurora genes family play an important throughout the entire cell cycle. In that Aurora B regulates chromosome segregation by ensuring the orientation of sister chromatids. As a consequence, the overexpression of Aurora B in diploid human cells NHDF induces the appearance of multinucleate cells. METHODS: Archive samples of normal and neoplastic prostate tissue, and prostate derived cell lines were screened for the expression of Aurora B. RESULTS: Immunohistochemical analysis showed increased nuclear expression of Aurora-B in high Gleason grade PCs respect to low and intermediate grade cases and in all cancers in respect to hyperplastic and normal glands. Furthermore, in the high Gleason grade anaplastic cancer tissues Aurora B expression was accompanied by the phosphorylation of the histone H3. In analogy to the in vivo situation, Aurora B was vigorously expressed in the androgen independent PC cell lines PC3 and DU145, while a very modest expression of the kinase was observed in the androgen sensitive LnCap cells and in the EPN cells, a line of epithelial cells derived from normal prostate tissue. In addition, in PC3 cells Aurora B expression is accompanied the by the phosphorylation of the histone H3. The block of Aurora B expression induced by an inhibitor of Aurora kinase activity significantly reduced the growth of prostate carcinoma cells, but not that of non-transformed EPN cells. CONCLUSIONS: Our data are the first demonstration of a role of Aurora B in PC progression. In addition, the observation that Aurora B specific inhibitors interfere with PC cell proliferation but not with that of non-transformed prostate epithelial cells suggest that Aurora B is a potential therapeutic target for PC

Emerging roles of ATF2 and the dynamic AP1 network in cancer

Cooperation among transcription factors is central for their ability to execute specific transcriptional programmes. The AP1 complex exemplifies a network of transcription factors that function in unison under normal circumstances and during the course of tumour development and progression. This Perspective summarizes our current understanding of the changes in members of the AP1 complex and the role of ATF2 as part of this complex in tumorigenesis.Fil: Lopez Bergami, Pablo Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: Lau, Eric . Burnham Institute for Medical Research; Estados UnidosFil: Ronai, Zeev . Burnham Institute for Medical Research; Estados Unido

Targeting TAO kinases using a new inhibitor compound delays mitosis and induces mitotic cell death in centrosome amplified breast cells

Thousand-and-one amino acid kinases (TAOKs) 1 and 2 are activated catalytically during mitosis and can contribute to mitotic cell rounding and spindle positioning. Here, we characterize a compound that inhibits TAOK1 and TAOK2 activity with IC50 values of 11-15 nM, is ATP-competitive and targets these kinases selectively. TAOK inhibition or depletion in centrosome amplified SKBR3 or BT549 breast cancer cell models increases the mitotic population, the percentages of mitotic cells displaying amplified centrosomes and multipolar spindles, induces cell death and inhibits cell growth. In contrast, non-tumorigenic and dividing bipolar MCF-10A breast cells appear less dependent on TAOK activity and can complete mitosis and proliferate in the presence of the TAOK inhibitor. We demonstrate that TAOK1 and TAOK2 localize to the cytoplasm and centrosomes respectively during mitosis. Live cell imaging shows that the TAOK inhibitor prolongs the duration of mitosis in SKBR3 cells, increases mitotic cell death and reduces the percentages of cells exiting mitosis, whereas MCF-10A cells continue to divide and proliferate. Over 80% of breast cancer tissues display supernumerary centrosomes and tumor cells frequently cluster extra centrosomes to avoid multipolar mitoses and associated cell death. Consequently, drugs that stimulate centrosome declustering and induce multipolarity are likely to target dividing centrosome amplified cancer cells preferentially, whilst sparing normal bipolar cells. Our results demonstrate that TAOK inhibition can enhance centrosome declustering and mitotic catastrophe in cancer cells and these proteins may therefore offer novel therapeutic targets suitable for drug inhibition and the potential treatment of breast cancers, where supernumerary centrosomes occur

High Proliferation Rate and a Compromised Spindle Assembly Checkpoint Confers Sensitivity to the MPS1 Inhibitor BOS172722 in Triple-Negative Breast Cancers

BOS172722 (CCT289346) is a highly potent, selective, and orally bioavailable inhibitor of spindle assembly checkpoint kinase MPS1. BOS172722 treatment alone induces significant sensitization to death, particularly in highly proliferative triple-negative breast cancer (TNBC) cell lines with compromised spindle assembly checkpoint activity. BOS172722 synergizes with paclitaxel to induce gross chromosomal segregation defects caused by MPS1 inhibitor-mediated abrogation of the mitotic delay induced by paclitaxel treatment. In in vivo pharmacodynamic experiments, BOS172722 potently inhibits the spindle assembly checkpoint induced by paclitaxel in human tumor xenograft models of TNBC, as measured by inhibition of the phosphorylation of histone H3 and the phosphorylation of the MPS1 substrate, KNL1. This mechanistic synergy results in significant in vivo efficacy, with robust tumor regressions observed for the combination of BOS172722 and paclitaxel versus either agent alone in long-term efficacy studies in multiple human tumor xenograft TNBC models, including a patient-derived xenograft and a systemic metastasis model. The current target indication for BOS172722 is TNBC, based on their high sensitivity to MPS1 inhibition, the well-defined clinical patient population with high unmet need, and the synergy observed with paclitaxel

Inhibition of Bromodomain Proteins Enhances Oncolytic HAdVC5 Replication and Efficacy in Pancreatic Ductal Adenocarcinoma (PDAC) Models.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of pancreatic cancer, which rapidly develops resistance to the current standard of care. Several oncolytic Human AdenoViruses (HAdVs) have been reported to re-sensitize drug-resistant cancer cells and in combination with chemotherapeutics attenuate solid tumour growth. Obstacles preventing greater clinical success are rapid hepatic elimination and limited viral replication and spread within the tumour microenvironment. We hypothesised that higher intratumoural levels of the virus could be achieved by altering cellular epigenetic regulation. Here we report on the screening of an enriched epigenetics small molecule library and validation of six compounds that increased viral gene expression and replication. The greatest effects were observed with three epigenetic inhibitors targeting bromodomain (BRD)-containing proteins. Specifically, BRD4 inhibitors enhanced the efficacy of Ad5 wild type, Ad∆∆, and Ad-3∆-A20T in 3-dimensional co-culture models of PDAC and in vivo xenografts. RNAseq analysis demonstrated that the inhibitors increased viral E1A expression, altered expression of cell cycle regulators and inflammatory factors, and attenuated expression levels of tumour cell oncogenes such as c-Myc and Myb. The data suggest that the tumour-selective Ad∆∆ and Ad-3∆-A20T combined with epigenetic inhibitors is a novel strategy for the treatment of PDAC by eliminating both cancer and associated stromal cells to pave the way for immune cell access even after systemic delivery of the virus

Metabolism of the dual FLT-3/Aurora kinase inhibitor CCT241736 in preclinical and human in vitro models: Implication for the choice of toxicology species.

CCT241736 is a dual fms-like tyrosine kinase 3 (FLT3)/Aurora kinase inhibitor in development for the treatment of acute myeloid leukaemia. The successful development of any new drug relies on adequate safety testing including preclinical toxicology studies. Selection of an appropriate preclinical species requires a thorough understanding of the compound's metabolic clearance and pathways, as well as other pharmacokinetic and pharmacodynamic considerations. In addition, elucidation of the metabolising enzymes in human facilitates improved clinical prediction based on population pharmacokinetics and can inform drug-drug interaction studies. Intrinsic clearance (CLint) determination and metabolite profiling of CCT241736 in human and four preclinical species (dog, minipig, rat and mouse) was undertaken in cryopreserved hepatocytes and liver microsomes. Recombinant human cytochrome P450 bactosomes (rCYP) were utilised to provide reaction phenotyping data and support prediction of metabolic pathways. CCT241736 exhibited low CLint in both hepatocytes and liver microsomes of human, dog, minipig and rat, but considerably higher CLint in mouse. CYP3A4 and CYP3A5 were identified as the major enzymes responsible for biotransformation of CCT241736 in human, exclusively forming five out of seven metabolites. Minipig showed greatest similarity to human with regard to both overall metabolic profile and abundance of specific metabolites relative to parent compound, and is therefore proposed as the most appropriate toxicological species. The greatest disparity was observed between human and dog. Based on metabolic profile, either mouse or rat is a suitable rodent species for toxicology studies