The location and development of Replicon Cluster Domains in early replicating DNA.

Background: It has been known for many years that in metazoan cells, replication origins are organised into clusters where origins within each cluster fire near-synchronously. Despite clusters being a fundamental organising principle of metazoan DNA replication, the genomic location of origin clusters has not been documented. Methods: We synchronised human U2OS by thymidine block and release followed by L-mimosine block and release to create a population of cells progressing into S phase with a high degree of synchrony. At different times after release into S phase, cells were pulsed with EdU; the EdU-labelled DNA was then pulled down, sequenced and mapped onto the human genome. Results: The early replicating DNA showed features at a range of scales. Wavelet analysis showed that the major feature of the early replicating DNA was at a size of 500 kb, consistent with clusters of replication origins. Over the first two hours of S phase, these Replicon Cluster Domains broadened in width, consistent with their being enlarged by the progression of replication forks at their outer boundaries. The total replication signal associated with each Replicon Cluster Domain varied considerably, and this variation was reproducible and conserved over time. We provide evidence that this variability in replication signal was at least in part caused by Replicon Cluster Domains being activated at different times in different cells in the population. We also provide evidence that adjacent clusters had a statistical preference for being activated in sequence across a group, consistent with the 'domino' model of replication focus activation order observed by microscopy. Conclusions: We show that early replicating DNA is organised into Replicon Cluster Domains that behave as expected of replicon clusters observed by DNA fibre analysis. The coordinated activation of different Replicon Cluster Domains can generate the replication timing programme by which the genome is duplicated.

Co-localisation of intra-nuclear membrane type-1 matrix metalloproteinase and hypoxia inducible factor-2α in osteosarcoma and prostate carcinoma cells.

Increased membrane type-1 matrix metalloproteinase (MT1-MMP) expression in osteosarcoma is predictive of poor prognosis and directs bone metastasis in prostate carcinoma. MT1-MMP subcellular localisation varies with oxygen tension, and, therefore, the aim of the present study was to assess protein interactions between MT1-MMP and the hypoxia inducible factors (HIF-1α and HIF-2α). MT1-MMP protein expression was investigated across a panel of cancer cell lines, including a positive and negative control. The hypoxia-induced alteration in subcellular location of MT1-MMP, HIF-1α and HIF-2α in the U2OS osteosarcoma cell line was assessed using subcellular fractionation. A proximity ligation assay was utilised to assess protein to protein interactions in the osteosarcoma U2OS and prostate carcinoma PC3 cell lines. U2OS and PC3 cells exhibited a significantly increased intra-nuclear interaction between MT1-MMP and HIF-2α in response to hypoxia. The role of this warrants further investigation as it may unveil novel opportunities to target MT1-MMP, which is of particular significance for osteosarcoma since current treatment options are limited.

The High-Affinity Interaction between ORC and DNA that Is Required for Replication Licensing Is Inhibited by 2-Arylquinolin-4-Amines.

In late mitosis and G1, origins of DNA replication must be "licensed" for use in the upcoming S phase by being encircled by double hexamers of the minichromosome maintenance proteins MCM2-7. A "licensing checkpoint" delays cells in G1 until sufficient origins have been licensed, but this checkpoint is lost in cancer cells. Inhibition of licensing can therefore kill cancer cells while only delaying normal cells in G1. In a high-throughput cell-based screen for licensing inhibitors we identified a family of 2-arylquinolin-4-amines, the most potent of which we call RL5a. The binding of the origin recognition complex (ORC) to origin DNA is the first step of the licensing reaction. We show that RL5a prevents ORC forming a tight complex with DNA that is required for MCM2-7 loading. Formation of this ORC-DNA complex requires ATP, and we show that RL5a inhibits ORC allosterically to mimic a lack of ATP.

Enhanced anti-tumour activity of the combination of the novel MEK inhibitor WX-554 and the novel PI3K inhibitor WX-037.

PURPOSE: Tumours frequently have defects in multiple oncogenic pathways, e.g. MAPK and PI3K signalling pathways, and combinations of targeted therapies may be required for optimal activity. This study evaluated the novel MEK inhibitor WX-554 and the novel PI3K inhibitor WX-037, as single agents and in combination, in colorectal carcinoma cell lines and tumour xenograft-bearing mice. METHODS: In vitro growth inhibition, survival and signal transduction were measured using the Sulforhodamine B, clonogenic and Western blotting assays, respectively, in HCT116 and HT29 cell lines. In vivo anti-tumour efficacy and pharmacokinetic properties were assessed in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice. RESULTS: The combination of WX-554 and WX-037 exhibited marked synergistic growth inhibition in vitro, which was associated with increased cytotoxicity and enhanced inhibition of ERK and S6 phosphorylation, compared to either agent alone. Pharmacokinetic analyses indicated that there was no PK interaction between the two drugs at low doses, but that at higher doses, WX-037 may delay the tumour uptake of WX-554. In vivo efficacy studies revealed that the combination of WX-037 and WX-554 was non-toxic and exhibited marked tumour growth inhibition greater than observed with either agent alone. CONCLUSION: These studies show for the first time that combination treatment with the novel MEK inhibitor WX-554 and the novel PI3K inhibitor WX-037 can induce synergistic growth inhibition in vitro, which translates into enhanced anti-tumour efficacy in vivo.

Combined PI3K and CDK2 inhibition induces cell death and enhances in vivo antitumour activity in colorectal cancer.

BACKGROUND: The phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway is commonly deregulated in human cancer, hence many PI3K and mTOR inhibitors have been developed and have now reached clinical trials. Similarly, CDKs have been investigated as cancer drug targets. METHODS: We have synthesised and characterised a series of 6-aminopyrimidines identified from a kinase screen that inhibit PI3K and/or mTOR and/or CDK2. Kinase inhibition, tumour cell growth, cell cycle distribution, cytotoxicity and signalling experiments were undertaken in HCT116 and HT29 colorectal cancer cell lines, and in vivo HT29 efficacy studies. RESULTS: 2,6-Diaminopyrimidines with an O(4)-cyclohexylmethyl substituent and a C-5-nitroso or cyano group (1,2,5) induced cell cycle phase alterations and were growth inhibitory (GI50<20 µM). Compound 1, but not 2 or 5, potently inhibits CDK2 (IC50=0.1 nM) as well as PI3K, and was cytotoxic at growth inhibitory concentrations. Consistent with kinase inhibition data, compound 1 reduced phospho-Rb and phospho-rS6 at GI50 concentrations. Combination of NU6102 (CDK2 inhibitor) and pictilisib (GDC-0941; pan-PI3K inhibitor) resulted in synergistic growth inhibition, and enhanced cytotoxicity in HT29 cells in vitro and HT29 tumour growth inhibition in vivo. CONCLUSIONS: These studies identified a novel series of mixed CDK2/PI3K inhibitors and demonstrate that dual targeting of CDK2 and PI3K can result in enhanced antitumour activity.

Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cells.

To prevent rereplication of genomic segments, the eukaryotic cell cycle is divided into two nonoverlapping phases. During late mitosis and G1 replication origins are "licensed" by loading MCM2-7 double hexamers and during S phase licensed replication origins activate to initiate bidirectional replication forks. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin-a "double fork stall" (DFS)-replication cannot be completed by conventional means. We previously showed how the distribution of replication origins in yeasts promotes complete genome replication even in the presence of irreversible fork stalling. This analysis predicts that DFSs are rare in yeasts but highly likely in large mammalian genomes. Here we show that complementary strand synthesis in early mitosis, ultrafine anaphase bridges, and G1-specific p53-binding protein 1 (53BP1) nuclear bodies provide a mechanism for resolving unreplicated DNA at DFSs in human cells. When origin number was experimentally altered, the number of these structures closely agreed with theoretical predictions of DFSs. The 53BP1 is preferentially bound to larger replicons, where the probability of DFSs is higher. Loss of 53BP1 caused hypersensitivity to licensing inhibition when replication origins were removed. These results provide a striking convergence of experimental and theoretical evidence that unreplicated DNA can pass through mitosis for resolution in the following cell cycle.

Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing.

The cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4-CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery-an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs.

Pre-clinical use of isogenic cell lines and tumours in vitro and in vivo for predictive biomarker discovery; impact of KRAS and PI3KCA mutation status on MEK inhibitor activity is model dependent.

Studies to identify predictive biomarkers can be carried out in isogenic cancer cell lines, which enable interrogation of the effect of a specific mutation. We assessed the effects of four drugs, the PI3K-mammalian target of rapamycin inhibitor dactolisib, the PI3K inhibitor pictrelisib, and the MEK (MAPK/ERK Kinase) inhibitors PD 0325901 and selumetinib, in isogenic DLD1 parental, KRAS(+/-), KRAS(G13D/-), PIK3CA(+/-) and PIK3CA(E545K/-) colorectal carcinoma cell lines. Importantly, we found substantial differences in the growth of these cells and in their drug sensitivity depending on whether they were studied under 2D (standard tissue culture on plastic) or 3D (in vitro soft agar and in vivo xenograft) conditions. DLD1 KRAS(+/-) and DLD1 PIK3CA(+/-) cells were more sensitive to MEK inhibitors than parental, DLD1 KRAS(G13D/-) and DLD1 PIK3CA(E545K/-) cells under 2D conditions, whereas DLD1 KRAS(G13D/-) and DLD1 PIK3CA(E545K/-) xenografts were sensitive to 10 mg/kg daily ×14 PD 0325901 in vivo (p ≤ 0.02) but tumours derived from parental DLD1 cells were not. These findings indicate that KRAS and PIK3CA mutations can influence the response of DLD1 colorectal cancer cell lines to MEK and PI3K inhibitors, but that the effect is dependent on the experimental model used to assess drug sensitivity.

The enhanced in vivo activity of the combination of a MEK and a PI3K inhibitor correlates with [18F]-FLT PET in human colorectal cancer xenograft tumour-bearing mice.

Combined targeting of the MAPK and PI3K signalling pathways in cancer may be necessary for optimal therapeutic activity. To support clinical studies of combination therapy, 3'-deoxy-3'-[(18)F]-fluorothymidine ([(18)F]-FLT) uptake measured by Positron Emission Tomography (PET) was evaluated as a non-invasive surrogate response biomarker in pre-clinical models. The in vivo anti-tumour efficacy and PK-PD properties of the MEK inhibitor PD 0325901 and the PI3K inhibitor GDC-0941, alone and in combination, were evaluated in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice, and [(18)F]-FLT PET investigated in mice bearing HCT116 xenografts. Dual targeting of PI3K and MEK induced marked tumour growth inhibition in vivo, and enhanced anti-tumour activity was predicted by [(18)F]-FLT PET scanning after 2 days of treatment. Pharmacodynamic analyses using the combination of the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901 revealed that increased efficacy is associated with an enhanced inhibition of the phosphorylation of ERK1/2, S6 and 4EBP1, compared to that observed with either single agent, and maintained inhibition of AKT phosphorylation. Pharmacokinetic studies indicated that there was no marked PK interaction between the two drugs. Together these results indicate that the combination of PI3K and MEK inhibitors can result in significant efficacy, and demonstrate for the first time that [(18)F]-FLT PET can be correlated to the improved efficacy of combined PI3K and MEK inhibitor treatment.