USP7 is essential for maintaining Rad18 stability and DNA damage tolerance.

Rad18 functions at the cross-roads of three different DNA damage response (DDR) pathways involved in protecting stressed replication forks: homologous recombination repair, DNA inter-strand cross-link repair and DNA damage tolerance. Although Rad18 serves to facilitate replication of damaged genomes by promoting translesion synthesis (TLS), this comes at a cost of potentially error-prone lesion bypass. In contrast, loss of Rad18-dependent TLS potentiates the collapse of stalled forks and leads to incomplete genome replication. Given the pivotal nature with which Rad18 governs the fine balance between replication fidelity and genome stability, Rad18 levels and activity have a major impact on genomic integrity. Here, we identify the de-ubiquitylating enzyme USP7 as a critical regulator of Rad18 protein levels. Loss of USP7 destabilizes Rad18 and compromises UV-induced PCNA mono-ubiquitylation and Pol η recruitment to stalled replication forks. USP7-depleted cells also fail to elongate nascent daughter strand DNA following UV irradiation and show reduced DNA damage tolerance. We demonstrate that USP7 associates with Rad18 directly via a consensus USP7-binding motif and can disassemble Rad18-dependent poly-ubiquitin chains both in vitro and in vivo. Taken together, these observations identify USP7 as a novel component of the cellular DDR involved in preserving the genome stability.

Assessment of p53 and ATM functionality in chronic lymphocytic leukemia by multiplex ligation-dependent probe amplification.

The ATM-p53 DNA-damage response (DDR) pathway has a crucial role in chemoresistance in CLL, as indicated by the adverse prognostic impact of genetic aberrations of TP53 and ATM. Identifying and distinguishing TP53 and ATM functional defects has become relevant as epigenetic and posttranscriptional dysregulation of the ATM/p53 axis is increasingly being recognized as the underlying cause of chemoresistance. Also, specific treatments sensitizing TP53- or ATM-deficient CLL cells are emerging. We therefore developed a new ATM-p53 functional assay with the aim to (i) identify and (ii) distinguish abnormalities of TP53 versus ATM and (iii) enable the identification of additional defects in the ATM-p53 pathway. Reversed transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) was used to measure ATM and/or p53-dependent genes at the RNA level following DNA damage using irradiation. Here, we showed that this assay is able to identify and distinguish three subgroups of CLL tumors (i.e., TP53-defective, ATM-defective and WT) and is also able to detect additional samples with a defective DDR, without molecular aberrations in TP53 and/or ATM. These findings make the ATM-p53 RT-MLPA functional assay a promising prognostic tool for predicting treatment responses in CLL.

The dual-acting chemotherapeutic agent Alchemix induces cell death independently of ATM and p53.

Topoisomerase inhibitors are in common use as chemotherapeutic agents although they can display reduced efficacy in chemotherapy-resistant tumours, which have inactivated DNA damage response (DDR) genes, such as ATM and TP53. Here, we characterise the cellular response to the dual-acting agent, Alchemix (ALX), which is a modified anthraquinone that functions as a topoisomerase inhibitor as well as an alkylating agent. We show that ALX induces a robust DDR at nano-molar concentrations and this is mediated primarily through ATR- and DNA-PK- but not ATM-dependent pathways, despite DNA double strand breaks being generated after prolonged exposure to the drug. Interestingly, exposure of epithelial tumour cell lines to ALX in vitro resulted in potent activation of the G2/M checkpoint, which after a prolonged arrest, was bypassed allowing cells to progress into mitosis where they ultimately died by mitotic catastrophe. We also observed effective killing of lymphoid tumour cell lines in vitro following exposure to ALX, although, in contrast, this tended to occur via activation of a p53-independent apoptotic pathway. Lastly, we validate the effectiveness of ALX as a chemotherapeutic agent in vivo by demonstrating its ability to cause a significant reduction in tumour cell growth, irrespective of TP53 status, using a mouse leukaemia xenograft model. Taken together, these data demonstrate that ALX, through its dual action as an alkylating agent and topoisomerase inhibitor, represents a novel anti-cancer agent that could be potentially used clinically to treat refractory or relapsed tumours, particularly those harbouring mutations in DDR genes.

The impact of SF3B1 mutations in CLL on the DNA-damage response.

Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage. Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction and apoptosis. In sole SF3B1 mutated cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was increased at baseline and upon irradiation. Our data demonstrate that single mutations in sf3b1 are associated with increased DNA damage and/or an aberrant response to DNA damage. Together, our observations may offer an explanation for the poor prognosis associated with SF3B1 mutations.

BET inhibition as a single or combined therapeutic approach in primary paediatric B-precursor acute lymphoblastic leukaemia.

Paediatric B-precursor ALL is a highly curable disease, however, treatment resistance in some patients and the long-term toxic effects of current therapies pose the need for more targeted therapeutic approaches. We addressed the cytotoxic effect of JQ1, a highly selective inhibitor against the transcriptional regulators, bromodomain and extra-terminal (BET) family of proteins, in paediatric ALL. We showed a potent in vitro cytotoxic response of a panel of primary ALL to JQ1, independent of their prognostic features but dependent on high MYC expression and coupled with transcriptional downregulation of multiple pro-survival pathways. In agreement with earlier studies, JQ1 induced cell cycle arrest. Here we show that BET inhibition also reduced c-Myc protein stability and suppressed progression of DNA replication forks in ALL cells. Consistent with c-Myc depletion and downregulation of pro-survival pathways JQ1 sensitised primary ALL samples to the classic ALL therapeutic agent dexamethasone. Finally, we demonstrated that JQ1 reduces ALL growth in ALL xenograft models, both as a single agent and in combination with dexamethasone. We conclude that targeting BET proteins should be considered as a new therapeutic strategy for the treatment of paediatric ALL and particularly those cases that exhibit suboptimal responses to standard treatment.

ZAP-70 is highly expressed in most cases of childhood pre-B cell acute lymphoblastic leukemia.

The tyrosine kinase ZAP-70 plays a critical role in signal transduction in T cells and NK cells but has limited expression in primary human B cells. ZAP-70 is, however, expressed in adult B cell chronic lymphocytic leukemia where it correlates with a poor prognosis. We wished to determine if ZAP-70 is also expressed in pediatric B cell malignancy. A quantitative PCR assay for ZAP-70 expression was established and ZAP-70 expression in a range of human B cell lines was compared with expression in the Jurkat T cell line. ZAP-70 expression was then determined in bone marrow lymphoblasts obtained from 12 patients with pre-B cell acute lymphoblastic leukemia (ALL). ZAP-70 expression was not detected in mature B cell lines but was detected in pre-B cell lines at a level comparable to that seen in T cells. ZAP-70 expression was strongly expressed in nine of the 12 cases of primary pre-B cell lymphoblastic leukemia. The T cell-associated protein kinase ZAP-70 is highly expressed in pre-B lineage cells and most cases of pre-B acute lymphoblastic leukemia. ZAP-70 expression may hold prognostic value for pre-B ALL and raises the prospect of a novel therapeutic target.

Down-regulation of ATM protein in HRS cells of nodular sclerosis Hodgkin's lymphoma in children occurs in the absence of ATM gene inactivation.

The tumour component of classical Hodgkin's lymphoma (cHL), Hodgkin Reed-Sternberg (HRS) cells, are believed to be derived from germinal centre (GC) B cells but intriguingly display a characteristic loss of B cell receptor (BCR) expression. The precise mechanisms by which BCR-negative HRS cell progenitors survive negative selection during the GC reaction remain obscure. Individuals with ataxia telangiectasia, caused by biallelic inactivation of the DNA damage response gene, ataxia telangiectasia mutated (ATM), have a higher risk of cHL development. Here we show that, in contrast to normal GC B cells that expressed low but detectable ATM protein, ATM protein was not detected in HRS cells of 17/18 cases of paediatric cHL, all but one with nodular sclerosis (NS) subtype. A comprehensive analysis of the ATM gene in microdissected HRS cells of nine representative tumours showed no evidence of either loss of heterozygosity or consistent pathogenic mutations. Furthermore, bisulphite sequencing of the ATM promoter from HRS cells of five tumours also revealed the absence of hypermethylation. Since our microarray data suggested significantly reduced ATM transcription in HRS cells compared to GC B cells, we conclude that loss of ATM expression could be the result of alterations in upstream regulators of ATM transcription. Importantly, ATM loss in paediatric cHLs has clinical implications and could be potentially exploited to guide future, less toxic, tumour-specific treatments.

Intraoperative cytology of clear cell carcinoma of the ovary.

OBJECTIVE: To describe the cytomorphology of clear cell carcinoma (CCC) of the ovary in intraoperative samples of peritoneal fluid, imprint and scraping samples of the tumour tissue. STUDY DESIGN: Fourteen histologically confirmed cases, stained by standard cytological procedures, were analysed by light microscopy. RESULTS: In 33.3% of peritoneal fluid samples and 92.9% of imprint and scraping cytological samples, besides variable clear cell cellular morphology, one or both distinct cytological characteristics were observed: eosinophilic, hyaline, extracellular, globular substance with or without formation of a 'raspberry' body and an eosinophilic, intracytoplasmic inclusions. These structures were clearly seen only in samples stained by May-Grünwald-Giemsa. CONCLUSION: Using cytological analysis of imprint and scraping samples of ovarian tumours it is possible to make a precise intraoperative cytological diagnosis in most cases of CCC of the ovary.

Morphometry and digital AgNOR analysis in cytological imprints of benign, borderline and malignant serous ovarian tumours.

AIM: The aim of the study was to determine values of a quantitative morphometry analysis of nuclear characteristics and argyrophilic nucleolar organizer regions (AgNORs) in differential cytodiagnosis of benign, atypically proliferating (borderline) and malignant serous ovarian tumours. METHODS: Cytological imprints of benign (n = 20), borderline (n = 19) and malignant (n = 20) ovarian serous tumours were analysed. A computerized, digital analysis was used to determine morphometric nuclear features, the number and characteristics of single AgNORs, cluster AgNORs, total AgNOR and AgNOR area/nucleus (relative area) ratio. According to their size AgNORs were classified in three categories. A one-way variance analysis and post hoc test (Scheffé) were used for statistical analysis. RESULTS: The morphometric nuclear analysis showed that benign, borderline and malignant serous ovarian tumours are statistically different (P < 0.001) according to the area and outline, the values being highest in malignant tumours and lowest in the borderline group. Digital analysis of AgNORs in benign, borderline and malignant groups showed that the total AgNOR number increases with progression of the lesion (meaning tumour malignancy) significantly (P < 0.001) between benign and malignant as well as between borderline and malignant serous ovarian tumours (P < 0.001). The progression of the lesion malignancy was accompanied by a significant (P < 0.001) progressive increase of the total and relative AgNOR area per nucleus. The AgNOR size increases from benign to malignant tumours and a statistically significant difference (P < 0.001) was observed in all three groups regarding small and large AgNORs. CONCLUSION: Combining different markers of morphometric nuclear characteristics and AgNOR values could improve differential cytodiagnosis of benign, borderline and malignant serous ovarian tumours.

Insights into the multistep transformation process of lymphomas: IgH-associated translocations and tumor suppressor gene mutations in clonally related composite Hodgkin's and non-Hodgkin's lymphomas.

Clonally related composite lymphomas of Hodgkin's lymphoma (HL) and Non-Hodgkin's lymphoma (NHL) represent models to study the multistep transformation process in tumorigenesis and the development of two distinct tumors from a shared precursor. We analyzed six such lymphomas for transforming events. The HLs were combined in two cases with follicular lymphoma (FL), and in one case each with B-cell chronic lymphocytic leukemia, splenic marginal zone lymphoma, mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). In the HL/FL and HL/MCL combinations, BCL2/IGH and CCND1/IGH translocations, respectively, were detected in both the HL and NHL. No mutations were found in the tumor suppressor genes FAS, NFKBIA and ATM. The HL/DLBCL case harbored clonal replacement mutations of the TP53 gene on both alleles exclusively in the DLBCL. In conclusion, we present the first examples of molecularly verified IgH-associated translocations in HL, which also show that BCL2/IGH or CCND1/IGH translocations can represent early steps in the pathogenesis of composite HL/FL or HL/MCL. The restriction of the TP53 mutations to the DLBCL in the HL/DLBCL case exemplifies a late transforming event that presumably happened in the germinal center and affected the fate of a common lymphoma precursor cell towards development of a DLBCL.

ATM mutations in sporadic lymphoid tumours.

Patients with the autosomal recessive disorder ataxia telangiectasia (A-T) show the biallelic inactivation of the ataxia telangiectasia mutated (ATM) gene. A-T patients exhibit a predisposition to the development of a wide range of lymphoid tumours, suggesting that the ATM protein normally plays an important role in the prevention of both T and B cell malignancies. The ATM protein is a 370 kDa protein kinase implicated in the integration of different cellular responses to particular forms of DNA damage. Several recent studies have reported the possibility that the ATM gene can act as a tumour suppressor gene in non A-T individuals. Frequent ATM inactivation was confirmed in three sporadic lymphoid tumours of mature phenotype: T cell prolymphocytic leukaemia (T-PLL), B-cell chronic lymphocytic leukaemia (B-CLL) and mantle cell lymphoma (MCL). Here, we provide a summary of the published ATM mutations in sporadic lymphoid tumours, including our own study on the role of ATM mutations in the pathogenesis of sporadic B-CLL. The published results suggest possible differences in the origin, the nature and distribution of ATM mutations between sporadic B-CLL, MCL and T-PLL. While ATM mutations in mature B cell tumours (B-CLL and MCL) represent a mixture of missense and truncating errors distributed across the whole of the ATM coding sequence, mutations in sporadic T-PLL appear to be predominantly missense, clustering in the region encoding the PI-3 kinase catalytic domain of the protein. The reason for this difference is unclear, but the difference itself supports the notion that the pathogenesis of B and T cell tumours on an ATM deficient background might be different. In addition, in both B-CLL and MCL ATM mutation carriers have been reported, raising the possibility that ATM mutation carriers may have an increased risk of developing these tumours. The existence as well as magnitude of the risk, however, remains to be established. Furthermore, our own studies indicate that the presence of ATM mutations in sporadic B-CLL causes a distinctive defect in response to DNA damaging agents, offering a possible explanation for the poor response of ATM mutant tumours to standard treatment. Therefore, one of the future challenges will be to devise strategies to bypass the existing defect in response to DNA damage and activate apoptosis in ATM mutant sporadic lymphoid tumours.

Molecular analysis of single colonies reveals a diverse origin of initial clonal proliferation in B-precursor acute lymphoblastic leukemia that can precede the t(12;21) translocation.

The pathogenesis of pediatric B-precursor acute lymphoblastic leukemia is largely unknown, and even with nonrandom chromosomal translocations present, the precise order of clonal molecular events is undefined. We developed an in vitro system using cytokines interleukin (IL)-3, IL-7, IL-10, and FMS-like tyrosine kinase 3 ligand with CD40 ligand-expressing fibroblasts to obtain single blast colonies from which clonal immunoglobulin heavy chain (IgH), T-cell receptor delta gene rearrangements, and, in t(12;21)-positive cases, TEL-AML1 fusion transcripts could be simultaneously PCR amplified. The proliferation of early tumor progenitors increased subclone detection enabling us, in seven diagnostic samples, to determine the stage of differentiation at which each leukemia occurred. Four were derived from the stage before initiation of IgH rearrangement, one during recombination of variable, joining, and diversity segments of the heavy chain gene VDJ(H), and two after completion of IgH rearrangement. Furthermore, analysis of a t(12;21)-positive leukemia with unusually late onset, identified both TEL-AML1-positive and -negative colonies carrying a clonal T-cell receptor delta rearrangement, inferring the presence of clonal expansion before the occurrence of the t(12;21). In contrast, in a typical, early onset t(12;21)-positive leukemia, the t(12;21) appeared to be the first clonal event. In both leukemias, the t(12;21) occurred before recombination of variable, joining and diversity segments of the heavy chain gene VDJ.

p53 dysfunction in B-cell chronic lymphocytic leukemia: inactivation of ATM as an alternative to TP53 mutation.

The well-established association between TP53 mutations and adverse clinical outcome in a range of human cancers reflects the importance of p53 protein in regulating tumor-cell growth and survival. Although it is theoretically possible for p53 dysfunction to arise through mechanisms that do not involve TP53 mutation, such a phenomenon has not previously been demonstrated in a sporadic tumor. Here, we show that p53 dysfunction in B-cell chronic lymphocytic leukemia (CLL) can occur in the absence of TP53 mutation and that such dysfunction is associated with mutation of the gene encoding ATM, a kinase implicated in p53 activation. Forty-three patients with CLL were examined for p53 dysfunction, as detected by impaired up-regulation of p53 and of the p53-dependent protein p21(CIP1/WAF1) after exposure to ionizing radiation (IR). Thirty (70%) patients had normal p53 responses and underwent progressive IR-induced apoptosis. In 13 (30%) patients, p21 up-regulation was markedly impaired, indicating p53 dysfunction. Six (14%) of these patients with p53 dysfunction had increased baseline levels of p53, were found to have TP53 mutations, and were completely resistant to IR-induced apoptosis. In the other 7 (16%) patients with p53 dysfunction, IR-induced p53 up-regulation and apoptosis were markedly impaired, but baseline levels of p53 were not increased, and no TP53 mutations were detected. Each of these patients was found to have at least one ATM mutation, and a variable reduction in ATM protein was detected in all 4 patients examined. This is the first study to provide a direct demonstration that p53 dysfunction can arise in a sporadic tumor by a mechanism that does not involve TP53 mutation. (Blood. 2001;98:814-822)

Residual ataxia telangiectasia mutated protein function in cells from ataxia telangiectasia patients, with 5762ins137 and 7271T-->G mutations, showing a less severe phenotype.

We have assessed several ataxia Telangiectasia mutated (ATM)-dependent functions in cells derived from ataxia telangiectasia patients, carrying either an ATM 5762ins137 splice site or a 7271T-->G missense mutation, with a less severe phenotype compared with the classical disorder. ATM kinase in vitro, from 5762ins137 cells, showed the same specific activity as ATM in normal cells, but the protein was present at low levels. In contrast, mutant ATM kinase activity in the 7271T-->G cells was only about 6% that of the activity in normal cells, although the level of mutant protein expressed was similar to normal cells. Phosphorylation of the DNA double strand break repair proteins Nbs1 and hMre11, following DNA damage, was observed in normal and 7271T-->G cells but was almost absent in both 5762ins137 and classical ataxia telangiectasia cells. The kinetics of p53 response was intermediate between normal and classical ataxia telangiectasia cells in both the 7271T-->G and 5762ins137 cells, but interestingly, c-Jun kinase activation following DNA damage was equally deficient in cell lines derived from all the ataxia telangiectasia patients. Our results indicate that levels of ATM kinase activity, but not induction of p53 or c-Jun kinase activity, in these cells correlate with the degree of neurological disorder in the patients.

hMRE11: genomic structure and a null mutation identified in a transcript protected from nonsense-mediated mRNA decay.

We showed recently that mutation of the hMRE11 gene identified a new ataxia telangiectasia-like disorder (ATLD). In this report we describe the genomic organization of the hMRE11 gene and the analysis of a promoter region that appears to direct the divergent transcription of hMRE11 and the adjacent gene. The characterization of the genomic organization of the hMRE11 gene allowed us to determine the basis of an apparent null hMRE11 allele present in the mother and two patients in one of our two ATLD families. Polymorphic markers in the hMRE11 gene, including the promoter region, provided evidence that the mutated maternal allele was not deleted. An exon by exon search revealed the presence of a missense mutation in exon 15, the effect of which was to create a premature termination codon. Transcripts derived from the mutant allele were found to be subject to nonsense-mediated mRNA decay (NMD). Therefore, this allele was effectively null, because little if any mRNA from it was available for translation. The ATLD patients carrying this protein-truncating hMRE11 mutation have survived because the null allele they inherited from their mother is present with a missense mutation inherited from their father, which is expressed as normal levels of partially functional MRE11 protein. The mutation in the maternal hMRE11 allele of family 2 was also identified in a further unrelated Italian family with ATLD and also found to be subject to NMD.