PDF file - 2.3MB, Supplementary Figures and Legends S1. Standstill mutants exhibit persistent growth defects and eventual embryonic death. S2. In situ hybridization of cdc25a in 36 hpf zebrafish embryos. S3. Knockdown of cdc25a by a translation-blocking morpholino phenocopies the standstill phenotype. S4. Zebrafish exhibit a robust cell-cycle arrest following treatment with ionizing radiation, as evaluated by anti-pH3 immunohistochemistry. S5. Inhibition of ATM with the ATM inhibitor KU55933 leads to radiosensitivity in zebrafish. S6. Inhibition of DNA damage response pathways with caffeine leads to increased cell proliferation in cdc25a-/- embryos. S7. The G1/S checkpoint is not activated in cdc25a-deficient embryos. S8. Loss of proliferation precedes the appearance of H2AX. S9. Overexpression of Cdc25A does not lead to decreased ATM function. S10. Deleterious effects of ATM knockdown.

Germ cell tumors (GCTs) are neoplasms of the testis, ovary and extragonadal sites that occur in infants, children, adolescents and adults. Post-pubertal (type II) malignant GCTs may present as seminoma, non-seminoma or mixed histologies. In contrast, pre-pubertal (type I) GCTs are limited to (benign) teratoma and (malignant) yolk sac tumor (YST). Epidemiologic and molecular data have shown that pre- and post-pubertal GCTs arise by distinct mechanisms. Dedicated studies of the genomic landscape of type I and II GCT in children and adolescents are lacking. Here we present an integrated genomic analysis of extracranial GCTs across the age spectrum from 0–24 years. Activation of the WNT pathway by somatic mutation, copy-number alteration, and differential promoter methylation is a prominent feature of GCTs in children, adolescents and young adults, and is associated with poor clinical outcomes. Significantly, we find that small molecule WNT inhibitors can suppress GCT cells both in vitro and in vivo. These results highlight the importance of WNT pathway signaling in GCTs across all ages and provide a foundation for future efforts to develop targeted therapies for these cancers.

Supplementary Table 1. Combined immunohistochemistry scores, yolk sac tumor and seminoma Supplementary Table 2. Immunohistochemistry scores for all tumors Supplementary Table 3. Sequences of primers used for quantitative RT-PCR Supplementary Figure 1. Complete blots of NCCIT and NTERA-2 cells treated with ligands. See Figure 4A for more details. Supplementary Figure 2. Complete blots of cells treated with rapamycin. See Figure 5 for more details. Supplementary Figure 3. Complete blots of NCCIT and NTERA-2 cells treated with erlotinib. See Figure 5 for more details. Supplementary Figure 4. Effect of EGFR and mTORC1 inhibitors on the viability of NCCIT cells. Supplementary Figure 5. Model depicting signaling pathways inhibited by erlotinib and rapamycin in NSGCTs

Many childhood Wilms tumors are driven by mutations in the microRNA biogenesis machinery, but the mechanism by which these mutations drive tumorigenesis is unknown. Here we show that the transcription factor pleomorphic adenoma gene 1 (PLAG1) is a microRNA target gene that is overexpressed in Wilms tumors with mutations in microRNA processing genes. Wilms tumors can also overexpress PLAG1 through copy number alterations, and PLAG1 expression correlates with prognosis in Wilms tumors. PLAG1 overexpression accelerates growth of Wilms tumor cells in vitro and induces neoplastic growth in the developing mouse kidney in vivo. In both settings, PLAG1 transactivates insulin-like growth factor 2 (IGF2), a key Wilms tumor oncogene, and drives mammalian target of rapamycin complex 1 (mTORC1) signaling. These data link microRNA impairment to the PLAG1-IGF2 pathway, providing new insight into the manner in which common Wilms tumor mutations drive disease pathogenesis.

Abstract Germ cell tumors (GCT) are malignant tumors that arise from pluripotent embryonic germ cells and occur in children and young adults. GCTs are treated with cisplatin-based regimens which, while overall effective, fail to cure all patients and cause significant adverse late effects. The seminoma and nonseminoma forms of GCT exhibit distinct differentiation states, clinical behavior, and response to treatment; however, the molecular mechanisms of GCT differentiation are not fully understood. We tested whether the activity of the mTORC1 and MAPK pathways were differentially active in the two classes of GCT. Here we show that nonseminomatous germ cell tumors (NSGCT, including embryonal carcinoma, yolk sac tumor, and choriocarcinoma) from both children and adults display activation of the mTORC1 pathway, while seminomas do not. In seminomas, high levels of REDD1 may negatively regulate mTORC1 activity. In NSGCTs, on the other hand, EGF and FGF2 ligands can stimulate mTORC1 and MAPK signaling, and members of the EGF and FGF receptor families are more highly expressed. Finally, proliferation of NSGCT cells in vitro and in vivo is significantly inhibited by combined treatment with the clinically available agents erlotinib and rapamycin, which target EGFR and mTORC1 signaling, respectively. These results provide an understanding of the signaling network that drives GCT growth and a rationale for therapeutic targeting of GCTs with agents that antagonize the EGFR and mTORC1 pathways. Mol Cancer Ther; 17(5); 1079–89. ©2018 AACR.

Wilms tumor is the most common pediatric kidney cancer and the fourth most common childhood cancer overall. Wilms tumor is treated with a combination of surgery, chemotherapy, and radiation, and, while most children are cured, patients suffer adverse late effects of therapy and survival remains poor in those with advanced-stage disease. Known driver mutations (in WT1 , WTX , and β-catenin) account for only one-third of Wilms tumor cases. To better define the genomic landscape of Wilms tumor, we performed whole-exome sequencing of 44 Wilms tumors to identify somatic mutations. Through this approach, we identified recurrent somatic mutations in MYCN , DROSHA , and DICER1 , which were mutually exclusive with known Wilms tumor driver mutations in WT1 and β-catenin. DROSHA and DICER1 somatic hotspot mutations occur at or near critical metal-binding residues in RNase III domains and partially or completely disrupt enzyme activity. Examination of miRNA expression in tumors, in vitro processing assays, and genomic editing in human cells demonstrate that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. While DICER1 RNase IIIB mutations preferentially impair processing of miRNAs that derive from the 5′ arm of pre-miRNA hairpins, DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Despite their distinct mechanisms of action, DROSHA and DICER1 mutations result in decreased expression of several common tumor-suppressing miRNAs, including multiple members of the let-7 and miR-15/16 families. We also identified a patient carrying a germline DICER1 loss-of-function allele who subsequently developed multiple malignancies with distinct DICER1 RNase IIIB somatic hotspot mutations, suggesting that DICER1 can act as both a tumor-suppressor and an oncogene depending on the nature of the mutation. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogram miRNA expression in human cancer and suggest that mutations causing impairment of miRNA processing define a distinct subclass of Wilms tumors. Citation Format: Kenneth S. Chen, Dinesh Rakheja, Yangjian Liu, Abhay A. Shukla, Joshua T. Mendell, James F. Amatruda. Mechanisms of tumorigenesis due to somatic mutations in DICER1 and DROSHA in childhood kidney cancers. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA12.

This study aims to identify novel therapeutic targets for nonseminomatous pediatric germ cell tumors (GCTs). GCTs are the most common cancer in young men, and affect both children and adolescents. They are histologically classified into two types: seminomatous GCTs (SGCTs), which are undifferentiated, and non-seminomatous GCTs (NSGCTs), which exhibit differentiation. Although cisplatin treatment is effective for many types of GCTs, cisplatin resistance, which is especially common in NSGCTs, confers poor prognosis for affected patients. However, because the signaling pathways and genes responsible for the development of different types of GCTs are not well-understood, few targeted therapies exist for GCTs, and no specific therapies exist for NSGCTs. Therefore, novel therapies to specifically target NSGCTs are needed. To determine a targetable pathway that is activated in GCTs, we used quantitative RT-PCR to measure the expression of growth factor receptors in pediatric GCTs, immunohistochemistry (IHC) on a panel of clinically annotated germ cell tumors, and Western blot as well as cell viability assays on NSGCT cell lines (NCCIT and NTERA-2) to determine the effect of inhibition of two signaling pathways on cell viability. Our RT-PCR results showed that multiple members of the EGF and FGF receptor families are expressed at higher levels in NSGCTs than SGCTs. In addition, we found that EGF and FGF2 stimulate Ras-MAPK as well as PI3K/mTOR signaling in NSGCT cell lines. Based on IHC staining of phosphorylated ERK1/2, mTOR, and S6 ribosomal protein, we showed that both the Ras-MAPK and PI3K-mTOR pathways are activated at higher levels in NSGCTs than SGCTs. The results suggested that inhibiting EGFR as well as mTORC1 may be effective in impairing the growth and survival of NSGCT cell lines. To test this hypothesis, we examined the effects of two small molecule inhibitors of EGFR and mTOR signaling, erlotinib and rapamycin, respectively, on the survival of NCCIT and NTERA-2 cell lines. We verified by Western blot that erlotinib inhibits components of the Ras-MAPK pathway in NSGCT cell lines, while rapamycin inhibits components of the PI3K-mTOR pathway. Cell survival experiments showed that while treatment with rapamycin or erlotinib alone decreased cell viability, sufficient reduction of survival could only be achieved with high concentrations that are not clinically feasible. However, a combination treatment of erlotinib and rapamycin synergistically inhibited the growth of the two NSGCT cell lines at clinically achievable concentrations. Our findings showed that NSGCTs are dependent on EGFR and mTOR signaling in vitro, and suggest that targeting these signaling pathways may be a promising therapy to specifically target chemoresistant NSGCTs. Citation Format: Albert Budhipramono, Dinesh Rakheja, Kenneth S. Chen, Nicholas Fustino, Abhay Shukla, Jonathan Wickiser, Theodore Laetsch, James F. Amatruda. EGFR and mTORC1 are novel therapeutic targets in nonseminomatous germ cell tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B03.

This study aims to identify novel therapeutic targets for nonseminomatous pediatric germ cell tumors (GCTs). GCTs are the most common cancer in young men, and affect both children and adolescents. They are histologically classified into two types: seminomatous GCTs (SGCTs), which are undifferentiated, and non-seminomatous GCTs (NSGCTs), which exhibit differentiation. Although cisplatin treatment is effective for many types of GCTs, cisplatin resistance, which is especially common in NSGCTs, confers poor prognosis for affected patients. However, because the signaling pathways and genes responsible for the development of different types of GCTs are not well-understood, few targeted therapies exist for GCTs, and no specific therapies exist for NSGCTs. Therefore, novel therapies to specifically target NSGCTs are needed. To determine a targetable pathway that is activated in GCTs, we used quantitative RT-PCR to measure the expression of growth factor receptors in pediatric GCTs, immunohistochemistry (IHC) on a panel of clinically annotated germ cell tumors, and Western blot as well as cell viability assays on NSGCT cell lines (NCCIT and NTERA-2) to determine the effect of inhibition of two signaling pathways on cell viability. Our RT-PCR results showed that multiple members of the EGF and FGF receptor families are expressed at higher levels in NSGCTs than SGCTs. In addition, we found that EGF and FGF2 stimulate Ras-MAPK as well as PI3K/mTOR signaling in NSGCT cell lines. Based on IHC staining of phosphorylated ERK1/2, mTOR, and S6 ribosomal protein, we showed that both the Ras-MAPK and PI3K-mTOR pathways are activated at higher levels in NSGCTs than SGCTs. The results suggested that inhibiting EGFR as well as mTORC1 may be effective in impairing the growth and survival of NSGCT cell lines. To test this hypothesis, we examined the effects of two small molecule inhibitors of EGFR and mTOR signaling, erlotinib and rapamycin, respectively, on the survival of NCCIT and NTERA-2 cell lines. We verified by Western blot that erlotinib inhibits components of the Ras-MAPK pathway in NSGCT cell lines, while rapamycin inhibits components of the PI3K-mTOR pathway. Cell survival experiments showed that while treatment with rapamycin or erlotinib alone decreased cell viability, sufficient reduction of survival could only be achieved with high concentrations that are not clinically feasible. However, a combination treatment of erlotinib and rapamycin synergistically inhibited the growth of the two NSGCT cell lines at clinically achievable concentrations. Our findings showed that NSGCTs are dependent on EGFR and mTOR signaling in vitro, and suggest that targeting these signaling pathways may be a promising therapy to specifically target chemoresistant NSGCTs. Citation Format: Albert Budhipramono, Dinesh Rakheja, Kenneth S. Chen, Nicholas Fustino, Abhay Shukla, Jonathan Wickiser, Theodore Laetsch, James F. Amatruda. EGFR and mTORC1 are novel therapeutic targets in nonseminomatous germ cell tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B03.

The single nucleotide val158met polymorphism in catechol o-methyltransferase (COMT) influences prefrontal cortex function. Working memory, dependent on the dorsolateral prefrontal cortex (DLPFC), has been repeatedly shown to be influenced by this COMT polymorphism. The high activity COMT val isoform is associated with lower synaptic dopamine levels. Altered synaptic dopamine levels are expected to lead to molecular adaptations within the synapse and within DLPFC neural circuitry. In this human post mortem study using high quality DLPFC tissue, we first examined the influence of the COMT val158met polymorphism on markers of dopamine neurotransmission, N-methyl-d-aspartate (NMDA) receptor subunits and glutamatic acid decarboxylase 67 (GAD67), all known to be critical to DLPFC circuitry and function. Next, we compared target gene expression profiles in a cohort of control and schizophrenia cases, each characterized by COMT genotype. We find that the COMT val allele in control subjects is associated with significant upregulation of GluN2A and GAD67 mRNA levels compared to met carriers. Comparisons between control and schizophrenia groups reveal that GluN2A, GAD67 and DRD2 are differentially regulated between diagnostic groups in a genotype specific manner. Chronic antipsychotic treatment in rodents did not explain these differences. These data demonstrate an association between COMTval158met genotype and gene expression profile in the DLPFC of controls, possibly adaptations to maintain DLPFC function. In schizophrenia val homozygotes, these adaptations are not seen and could reflect pathophysiologic mechanisms related to the known poorer performance of these subjects on DLPFC-dependent tasks.

Abstract Wilms tumor is the most common pediatric kidney cancer and the fourth most common childhood cancer overall. It is treated with a combination of surgery, chemotherapy, and radiation, and while most children are cured, survival remains poor in those with advanced-stage disease. Known driver mutations (in WT1, WTX, and CTNNB1) account for only one-third of Wilms tumor cases. To better define the genomic landscape of Wilms tumor, we performed whole-exome sequencing of 44 Wilms tumors. We also sequenced matched germline DNA by whole-exome sequencing (15 cases) or targeted Sanger sequencing (29 cases) to identify somatic mutations. Through this approach, we identified recurrent somatic mutations in MYCN, DROSHA, and DICER1, which were mutually exclusive with known Wilms tumor mutations in WT1 and CTNNB1. The ribonucleases DROSHA and DICER1 perform two key steps in the biogenesis of microRNAs (miRNAs), which are ∼22-nt non-coding RNAs that regulate the stability and translation of target mRNAs. DROSHA and DICER1 mutations occurred at or near conserved metal-binding residues in their ribonuclease (RNase) IIIB domains. In vitro processing assays modeling these mutations showed that they partially or completely disrupted the ribonuclease activity of DROSHA and DICER1. In addition, next-generation sequencing of small RNAs in these tumors revealed that the DICER1 mutations preferentially impaired processing of miRNAs derived from the 5′ arm of pre-miRNA hairpins, including the let-7 tumor suppressor miRNA family. Processing of 5′-derived miRNAs is specifically dependent on DICER1 RNase IIIB activity. Although DROSHA-mutant tumors did not exhibit skewed expression of 5′ vs. 3′-derived miRNAs, expression of let-7 and other putative tumor suppressor miRNAs was reduced in these tumors as well. While germline and somatic mutations in DICER1 have been reported in some cancers, this is the first report of recurrent DROSHA mutations in any human tumor type. Moreover, these results suggest that impairment of miRNA processing by somatic mutation of DROSHA and DICER1 defines a novel subclass of Wilms tumor. Citation Format: Dinesh Rakheja, Kenneth S. Chen, Yangjian Liu, Abhay A. Shukla, Sara Hildebrand, Vanessa Schmid, Xiaoyong Sun, Xin Feng, Tsung-Cheng Chang, Shama Khokhar, Nitin J. Karandikar, James S. Malter, Joshua T. Mendell, James F. Amatruda. Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A22.

5-Lipoxygenase (5-LO) is one of the members of Lipoxygenase family. It breaks down arachidonic acid to pro-inflammatory compounds like leukotrienes. Leukotriene plays a major role in the inflammatory process. In this study, while cloning full length 5-LO, a novel splice variant of 5-LO (t5-LO) was found to be expressed in HepG2 cell line. The complete ORF of t5-LO is 420 bp long, expressing 139 amino acid long proteins from N-terminal. The splice variant of 5-LO was cloned, expressed, purified in bacterial system and characterized by MS/MS and western blot experiments. The full length 5-LO is 674 amino acids long encoded by 2,025 bp long ORF. RT-PCR and western blot revealed that t5-LO is extensively expressed in HepG2 cell line.

Wilms tumour is the most common childhood kidney cancer. Here we report the whole-exome sequencing of 44 Wilms tumours, identifying missense mutations in the microRNA (miRNA)-processing enzymes DROSHA and DICER1, and novel mutations in MYCN, SMARCA4 and ARID1A. Examination of tumour miRNA expression, in vitro processing assays and genomic editing in human cells demonstrates that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. DICER1 RNase IIIB mutations preferentially impair processing of miRNAs deriving from the 5'-arm of pre-miRNA hairpins, while DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Both DROSHA and DICER1 mutations impair expression of tumour-suppressing miRNAs, including the let-7 family, important regulators of MYCN, LIN28 and other Wilms tumour oncogenes. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogramme miRNA expression in human cancer and suggest that these defects define a distinct subclass of Wilms tumours.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Wilms Tumor is the most common pediatric cancer of the genitourinary tract. It is a primitive multilineage malignant neoplasm of embryonic renal precursor cells and is often associated with persistent foci of embryonic renal tissue known as Nephrogenic Rests. However, early molecular events that orchestrate the progression of Nephrogenic Rests to Wilms Tumor are unknown. Protein tyrosine phosphorylation, a critical regulator of signaling pathways, is a reversible process controlled by the opposing activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). The possible role of PTPs in Wilms Tumor has not been explored. Through immunohistochemical (IHC) analysis of tissue microarrays containing Wilms Tumors, Nephrogenic Rests, and normal kidney, we have identified downregulation of protein tyrosine phosphatase epsilon (PTPE) as a potential early event in Wilms tumorigenesis. IHC results showed strong staining for PTPE in 6 of 6 (100%) Nephrogenic Rests. In contrast, of the 51 Wilms Tumors studied, PTPE staining was absent in 2 (4%), weak in 26 (51%), moderate in 16 (31%), and strong in 7 (14%) compared to normal renal cortex. In addition, quantitative reverse-transcription PCR showed decreased PTPE expression in Wilms Tumor tissues compared to normal renal cortex. To identify potential targets of PTPE, we performed phosphokinase arrays and demonstrated that overexpression of PTPE in HEK293 cells results in dephosphorylation of Focal adhesion kinase (FAK), Lymphocyte-specific protein tyrosine kinase (LCK), and Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog (FGR). To validate the relevance of PTPE in Wilms Tumors, we established primary tumor cultures from nephrectomy specimens of children undergoing treatment for Wilms Tumor. Consistent with our IHC data, these primary cultures exhibit varying levels of PTPE expression. We have extended our results in these primary Wilms Tumor cells and found that overexpression of PTPE leads to dephosphorylation of FAK and LCK. We also probed downstream effector pathways and found that PTPE overexpression leads to dephosphorylation and inactivation of AKT (T308 and S375) and mTOR (S2448). Collectively, these PTPE targets have been established to control essential functions like cell proliferation, adhesion, and migration. In light of our results, we suggest that PTPE is a putative tumor suppressor in Wilms Tumor, acting to control signaling through FAK, LCK, and other SRC family proteins, as well as the PI3K/AKT/mTOR pathway. These results establish the utility of primary Wilms Tumor culture models, and provide the rationale for exploring SRC family kinase and AKT/mTOR pathway inhibitors as novel targeted agents for the treatment of Wilms Tumor. Citation Format: Abhay A. Shukla, Shama Khokhar, James F. Amatruda, Dinesh Rakheja. Protein tyrosine phosphatase epsilon (PTPE), a candidate tumor-suppressor in Wilms tumors of childhood, can regulate PI3K/AKT/mTOR pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5046. doi:10.1158/1538-7445.AM2013-5046

Human breast cancer cell proliferation involves a complex interaction between growth factors, steroid hormones and peptide hormones. The interaction of growth factors, such as epidermal growth factor (EGF), with their receptors on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acid such as arachidonic acid, which can be further metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) pathways to produce prostaglandins. The high concentration of prostaglandins has been associated with chronic inflammatory diseases and several types of human cancers. This is due to the over expression COX, LOX and other inflammatory enzymes. Ten peptides were designed and synthesized by solid phase peptide synthesis and analyzed in vitro for enzyme inhibition. Out of these peptides, YWCS had shown significant inhibitory effects. The dissociation constant (K(D)) was determined by surface plasmon resonance (SPR) analysis and was found to be 3.39 × 10(-8) M and 8.6 × 10(-8) M for YWCS and baicalein (positive control), respectively. The kinetic constant Ki was 72.45 × 10(-7) M as determined by kinetic assay. The peptide significantly reduced the cell viability of estrogen positive MCF-7 and estrogen negative MDA-MB-231 cell line with the half maximal concentration (IC(50)) of 75 µM and 400 µM, respectively. The peptide also induced 49.8% and 20.8% apoptosis in breast cancer cells MCF-7 and MDA-MB-231, respectively. The YWCS was also found to be least hemolytic at a concentration of 358 µM. In vivo studies had shown that the peptide significantly inhibits tumor growth in mice (p<0.017). This peptide can be used as a lead compound and complement for ongoing efforts to develop differentiation therapies for breast cancer.

Abstract The early development of vertebrate embryos is characterized by rapid cell proliferation necessary to support the embryo's growth. During this period, the embryo must maintain a balance between ongoing cell proliferation and mechanisms that arrest or delay the cell cycle to repair oxidative damage and other genotoxic stresses. The ataxia-telangiectasia mutated (ATM) kinase is a critical regulator of the response to DNA damage, acting through downstream effectors, such as p53 and checkpoint kinases (CHK) to mediate cell-cycle checkpoints in the presence of DNA damage. Mice and humans with inactivating mutations in ATM are viable but have increased susceptibility to cancers. The possible role of ATM in limiting cell proliferation in early embryos has not been fully defined. One target of ATM and CHKs is the Cdc25 phosphatase, which facilitates cell-cycle progression by removing inhibitory phosphates from cyclin-dependent kinases (CDK). We have identified a zebrafish mutant, standstill, with an inactivating mutation in cdc25a. Loss of cdc25a in the zebrafish leads to accumulation of cells in late G2 phase. We find that the novel family member cdc25d is essential for early development in the absence of cdc25a, establishing for the first time that cdc25d is active in vivo in zebrafish. Surprisingly, we find that cell-cycle progression in cdc25a mutants can be rescued by chemical or genetic inhibition of ATM. Checkpoint activation in cdc25a mutants occurs despite the absence of increased DNA damage, highlighting the role of Cdc25 proteins to balance constitutive ATM activity during early embryonic development. Mol Cancer Res; 10(11); 1451–61. ©2012 AACR.

The expression and significance of cathepsin L (CTSL) has been extensively studied in solid tumours. However no such information in chronic myeloid leukaemia (CML) was available. We investigated the activity and expression of this protease in peripheral blood mononuclear cells (PBMCs) of 47 adult CML patients. Thirty adults suffering from systemic diseases and 50 healthy volunteers served as controls. The mRNA levels of CTSL, its specific endogenous inhibitor cystatin C and transcriptional up‐regulator vascular endothelial growth factor (VEGF) were quantitated by real‐time qPCR. CTSL protease activity and its mRNA expression were significantly higher in CML chronic phase (CP) patients compared to CML accelerated phase/blast crisis (AP/BC) patients and controls (P≤ 0.001). VEGF whose expression was most pronounced in CP and declined (P≤ 0.001) in the advanced phases of the malignancy exhibited a strong positive correlation with CTSL expression (r= 0.97; P≤ 0.001). Cystatin C expression was significantly lower (P≤ 0.001) in CML and displayed inverse correlation with CTSL (r=−0.713; P≤ 0.001) activity. CTSL promoter was significantly hypomethylated in CML CP compared to CML AP/BC patients as well as controls. K562, a BC CML cell line displayed CTSL activity, expression and methylation status of CTSL promoter that was comparable to CML AP/BC patients. Treatment of these cells or PBMCs isolated from CML AP/BC patients with 5′‐aza‐cytidine resulted in a dramatic increase in CSTL activity and/or expression thereby demonstrating the role of promoter methylation in the stage specific expression of CTSL in CML. Differential expression of CTSL in CML at various stages of malignancy may prove useful in identification of the high‐risk patients thereby facilitating better management of disease.