p53, stem cell biology and childhood blastomas.

PURPOSE OF REVIEW: Childhood blastomas, unlike adult cancers, originate from developing organs in which molecular and cellular features exhibit differentiation arrest and embryonic characteristics. Conventional cancer therapies, which rely on the generalized cytotoxic effect on rapidly dividing cells, may damage delicate organs in young children, leading to multiple late effects. Deep understanding of the biology of embryonal cancers is crucial in reshaping the cancer treatment paradigm for children. RECENT FINDINGS: p53 plays a major physiological role in embryonic development, by controlling cell proliferation, differentiation and responses to cellular stress. Tumor suppressor function of p53 is commonly lost in adult cancers through genetic alterations. However, both somatic and germline p53 mutations are rare in childhood blastomas, suggesting that in these cancers, p53 may be inactivated through other mechanisms than mutation. In this review, we summarize current knowledge about p53 pathway inactivation in childhood blastomas (specifically neuroblastoma, retinoblastoma and Wilms' tumor) through various upstream mechanisms. Laboratory evidence and clinical trials of targeted therapies specific to exploiting p53 upstream regulators are discussed. SUMMARY: Despite the low rate of inherent TP53 mutations, p53 pathway inactivation is a common denominator in childhood blastomas. Exploiting p53 and its regulators is likely to translate into more effective targeted therapies with minimal late effects for children. (see Video Abstract, Supplemental Digital Content 1, http://links.lww.com/COON/A23).

Combined ATR and DNA-PK Inhibition Radiosensitizes Tumor Cells Independently of Their p53 Status.

Head and neck squamous cell carcinoma (HNSCC) is a significant cause of cancer deaths. Cisplatin-based chemoradiotherapy is a standard of care for locally advanced disease. ATR and DNA-PK inhibition (DNA-PKi) are actively being investigated in clinical trials with preclinical data supporting clinical translation as radiosensitizers. Here, we hypothesized that targeting both ATR and DNA-PK with small molecule inhibitors would increase radiosensitization of HNSCC cell lines. Radiosensitization was assessed by Bliss independence analysis of colony survival data. Strong cell cycle perturbing effects were observed with ATR inhibition reversing the G2/M arrest observed for radiation-DNA-PKi. Increased apoptosis in combination groups was measured by Sub-G1 DNA populations. DNA-PKi increased radiation-induced RAD51 and gamma-H2Ax foci, with the addition of ATR inhibition reducing levels of both. A sharp increase in nuclear fragmentation after aberrant mitotic transit appears to be the main driver of decreased survival due to irradiation and dual ATR/DNA-PKi. Dual inhibition of DNA-PK and ATR represents a novel approach in combination with radiation, with efficacy appearing to be independent of p53 status. Due to toxicity concerns, careful assessment is necessary in any future translation of single or dual radiosensitization approaches. Ongoing clinical trials into the ATR inhibitor AZD6738 plus radiation, and the phenotypically similar combination of AZD6738 and the PARP inhibitor olaparib, are likely to be key in ascertaining the toxicity profile of such combinations.

An orally bioavailable Chk1 inhibitor, CCT244747, sensitizes bladder and head and neck cancer cell lines to radiation.

PURPOSE: Chk1 inhibition increases cell sensitivity to both chemotherapy and radiotherapy in several tumour types and is, therefore, a promising anti-cancer approach. Although several Chk1 inhibitors have been developed, their clinical progress has been hampered by low bioavailability and off-target toxicities. MATERIALS AND METHODS: We characterized the radiosensitizing activity of CCT244747, the first orally bioavailable Chk1 inhibitor. We used a panel of bladder and head and neck cancer cell lines and monitored the effect of combining CCT244747 with radiation both in in vitro and in vivo models. RESULTS: CCT244747 sensitized cancer cell lines to radiation in vitro and resulted in a growth delay in cancer xenograft models associated with a survival benefit. Radiosensitization was elicited by abrogation of the radiation-induced G2 arrest and premature entry into mitosis. CONCLUSIONS: CCT244747 is a potent and specific Chk1 inhibitor that can be administered orally. It radiosensitizes tumour cell lines and represents a new therapy for clinical application in combination with radiotherapy.

Radiosensitization by the ATR Inhibitor AZD6738 through Generation of Acentric Micronuclei.

AZD6738 is an orally active ATR inhibitor (ATRi) currently in phase I clinical trials. We found in vitro growth inhibitory activity of this ATRi in a panel of human cancer cell lines. We demonstrated radiosensitization by AZD6738 to single radiation fractions in multiple cancer cell lines independent of both p53 and BRCA2 status by the clonogenic assay. Radiosensitization by AZD6738 to clinically relevant doses of fractionated radiation was demonstrated in vitro using a 3D tumor spheroid model and, in vivo, AZD6738 radiosensitized by abrogating the radiation-induced G2 cell-cycle checkpoint and inhibiting homologous recombination. Mitosis with damaged DNA resulted in mitotic catastrophe as measured by micronucleus formation by live-cell fluorescent-ubiquitination cell-cycle imaging of cell-cycle progression and nuclear morphology. Induction of micronuclei was significantly more prominent for AZD6738 compared with inhibition of the downstream kinase CHK1 alone at isoeffective doses. Micronuclei were characterized as acentric chromosomal fragments, which displayed characteristics of increased DNA damage and cell-cycle dyssynchrony when compared with the primary nucleus. Mol Cancer Ther; 16(1); 25-34. ©2016 AACR.

Molecular analysis in a GALNS study cohort of 15 Tunisian patients: description of a novel mutation.

BACKGROUND: Mucopolysaccharidosis type IVA (MPS IVA) is an autosomal recessive disease caused by the deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The purpose of this study was to analyze the GALNS mutations and the haplotypes associated. METHODS: Mutation screening of the GALNS gene was performed by direct sequence analysis using DNA samples from 15 unrelated Tunisian MPS IVA patients. We also analyzed the haplotypes associated with the novel mutation and with the other reported GALNS mutations. RESULTS: We have identified an unreported missense mutation p.D288G (c.863A > G) in one patient, the most frequently c.120 + 1G > A (IVS1 + 1G > A) mutation in eleven MPS IVA patients and three previously reported mutations p.G66R, p.A85T and p.R386C on the other MPS IVA patients. All the studied patients were homozygous for these identified mutations. Bioinformatics analysis predicted the novel mutation as being probably pathogenic. These findings with the unobserved p.D288G mutation in controls subjects, suggested that it is a disease-causing mutation, which was correlated with the severe phenotype observed in the patients. We have found that the two GALNS unreported and reported mutations, respectively p.D288G and p.R386C, were associated with a common and specific haplotype. CONCLUSION: Our results were in agreement with previous reports from Tunisia, suggesting, on one hand the genotype/phenotype correlations in MPS IVA patients and the other hand the haplotype analyses were useful for determination of mutation origin in Tunisian population.

Trametinib radiosensitises RAS- and BRAF-mutated melanoma by perturbing cell cycle and inducing senescence.

PURPOSE: Radiotherapy (RT) is used frequently in patients with melanoma, but results are suboptimal because the disease is often radioresistant. This may be due to constitutive activation of MAPK pathway signalling through mutations involving RAS/RAF. Thus, we studied whether trametinib, a potent and selective allosteric inhibitor of MEK1/2 could improve the efficacy of RT. METHODS AND MATERIALS: Clonogenic survival assays were performed in human BRAF-mutant (A375), NRAS-mutant (D04, WM1631), KRAS-mutant (WM1791c) and wild-type (PMWK) melanoma cell lines. The effects of trametinib with and without radiation on protein levels of MEK effectors were measured by immunoblot analyses. Cell cycle effects, DNA damage repair, mitotic catastrophe and senescence were measured using flow cytometry, γH2Ax staining, nuclear fragmentation and ß-galactosidase staining, respectively. Additionally, athymic mice with D04 flank tumours were treated with fractionated RT after gavage with trametinib and monitored for tumour growth. RESULTS: All cell lines, except PMWK, exhibited enhanced cytotoxicity when RT was combined with trametinib compared to either agent alone. Sensitiser enhancement ratios were 1.70, 1.32, 1.10, and 1.70 for A375, D04, WM1361 and WM1791c, respectively. Trametinib efficiently blocked RT-induced phosphorylation of ERK at nanomolar concentrations. Increased radiosensitivity correlated with prolonged G1 arrest and reduction in the radioresistant S phase up to 48 h following RT. A larger population of senescence-activated ß-galactosidase-positive cells was seen in the trametinib pretreated group, and this correlated with activation of two of the major mediators of induced senescence, p53 and pRb. Mice receiving the combination treatment (trametinib 1mg/kg and RT over 3 days) showed a reduced mean tumour volume compared with mice receiving trametinib alone (p=0.016), or RT alone (p=0.047). No overt signs of drug toxicity were observed. CONCLUSION: Trametinib radiosensitised RAS-/RAF-mutated melanoma cells by inducing prolonged G1 arrest and premature senescence. In this pre-clinical study we demonstrate that combining trametinib and RT is well tolerated, and reduces tumour growth in vivo.

Effects of Δ40p53, an isoform of p53 lacking the N-terminus, on transactivation capacity of the tumor suppressor protein p53.

BACKGROUND: The p53 protein is expressed as multiple isoforms that differ in their N- and C-terminus due to alternative splicing, promoter or codon initiation usage. Δ40p53 lacks the first 39 residues containing the main transcriptional activation domain, resulting from initiation of translation at AUG +40 in fully spliced p53 mRNA or in a specific variant mRNA retaining intron 2. Overexpression of Δ40p53 antagonizes wild-type p53 in vitro. However, animal models of Δ40p53 in mouse or Zebrafish have shown complex phenotypes suggestive of p53-dependent growth suppressive effects. METHODS: We have co-transfected expression vectors for p53 and Δ40p53 in p53-null cell lines Saos-2 and H1299 to show that Δ40p53 forms mixed oligomers with p53 that bind to DNA and modulate the transcription of a generic p53-dependent reporter gene. RESULTS: In H1299 cells, co-expression of the two proteins induced a decrease in transcription with amplitude that depended upon the predicted composition of the hetero-tetramer. In Saos-2, a paradoxical effect was observed, with a small increase in activity for hetero-tetramers predicted to contain 1 or 2 monomers of Δ40p53 and a decrease at higher Δ40p53/p53 ratios. In this cell line, co-transfection of Δ40p53 prevented Hdm2-mediated degradation of p53. CONCLUSION: Δ40p53 modulates transcriptional activity by interfering with the binding of Hdm2 to hetero-tetramers containing both Δ40p53 and p53. These results provide a basis for growth suppressive effects in animal models co-expressing roughly similar levels of p53 and Δ40p53.

Redox control and interplay between p53 isoforms: roles in the regulation of basal p53 levels, cell fate, and senescence.

The p53 tumor suppressor protein has achieved stardom in molecular oncology owing to frequent inactivation in a large range of cancers. Known as a factor activated by multiple forms of stress and causing a broad suppressive response to DNA damage, its regulation and functions in basal (non-stress) conditions has received relatively little attention. We summarize recent findings highlighting roles of p53 in physiological processes such as stem cell maintenance, development, aging and senescence, and regulation of basal oxidative cell metabolism. We suggest that these properties are regulated through two integrated biochemical systems: the redox-sensing capacity of the p53 protein (due to its structural features and its regulation by redox factors such as thioredoxin, metallothioneins, or the redox-repair enzyme APE1/ref-1), and the expression of p53 as multiple isoforms with antagonist effects. We propose that interactions between p53 and its isoforms Δ40p53 or Δ133p53 play critical roles in intracellular signaling by reactive oxygen species. We also discuss evidence that p53 controls energy production by repressing glycolysis and enhancing mitochondrial oxidative metabolism. Together, these mechanisms suggest that p53 acts not only as a "guardian of the genome" against DNA damage but also as a finely-tuned regulator of redox-dependent physiological processes.