Cyclin C was cloned as a growth-promoting G1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumor suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an “orphan” CDK19 kinase, as well as CDK8 and CDK3. These cyclin C-CDK complexes phosphorylate Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin C-knockout mice. Cyclin C ablation or heterozygosity collaborate with other oncogenic lesions and accelerate development of T-cell-acute lymphoblastic leukemia (T-ALL). Furthermore, the cyclin C gene is heterozygously deleted in a significant fraction of human T-ALL, and these tumors express reduced cyclin C levels. We also describe point mutations in human T-ALL that render cyclin C-CDK unable to phosphorylate ICN1. Hence, tumor cells may develop different strategies to evade cyclin C inhibitory function.

SummaryWe investigated the outcome for children and young people with Early T-precursor acute lymphoblastic leukaemia (ETP-ALL), a recently described poor prognosis subgroup of T-ALL, treated on a contemporary protocol, UKALL 2003. After a median follow-up of 4 years and 10 months, the ETP sub-group, representing 16% of T-ALL patients, had non-significantly inferior 5-year event-free survival (76Á7% vs. 84Á6%, P = 0Á2) and overall survival (82Á4% vs. 90Á9%, P = 0Á1), and a higher relapse rate (18Á6% vs. 9Á6%, P = 0Á1) compared to typical T-ALL. ETP-ALL has an intermediate risk outcome, which does not warrant experimental treatment or first remission allogeneic transplant for the group universally.

Acquiring a mechanistic understanding of the processes underlying the renal clearance of drug molecules in man has been hampered by a lack of robust in vitro models of human proximal tubules. Several human renal epithelial cell lines derived from the renal cortex are available, but few have been characterised in detail in terms of transporter expression. This includes the HK-2 proximal tubule cell line, which has been used extensively as a model of nephrotoxicity. The aim of this study was to investigate the expression and function of drug transporters in HK-2 cells and their suitability as an in vitro model of the human proximal tubule. qPCR showed no mRNA expression of the SLC22 transporter family (OAT1, OAT3, OCT2) in HK-2 cells compared to renal cortex samples. In contrast, SLC16A1 (MCT1), which is important in the uptake of monocarboxylates, and SLCO4C1 (OATP4C1) were expressed in HK-2 cells. The functional expression of these transporters was confirmed by uptake studies using radiolabelled prototypic substrates DL-lactate and digoxin, respectively. The mRNA expression of apical membrane efflux transporters ABCB1 (MDR1) and several members of the ABCC family (multidrug resistance proteins, MRPs) was shown by qPCR. ABCG1 (BCRP) was not detected. The efflux of Hoechst 33342, a substrate for MDR1, was blocked by MDR1 inhibitor cyclosporin A, suggesting the functional expression of this transporter. Similarly, the efflux of the MRP-specific fluorescent dye glutathione methylfluorescein was inhibited by the MRP inhibitor MK571. Taken together, the results of this study suggest that HK-2 cells are of limited value as an in vitro model of drug transporter expression in the human proximal tubule.

The molecular mechanisms involved in disease progression and relapse in Tcell acute lymphoblastic leukemia (T-ALL) are poorly understood. We used single nucleotide polymorphism array analysis to analyze paired diagnostic and relapsed T-ALL samples to identify recurrent genetic alterations in T-ALL. This analysis showed that diagnosis and relapsed cases have common genetic alterations, but also that relapsed samples frequently lose chromosomal markers present at diagnosis, suggesting that relapsed T-ALL emerges from an ancestral clone different from the major leukemic population at diagnosis. In addition, we identified deletions and associated mutations in the WT1 tumor suppressor gene in 2 of 9 samples. Subsequent analysis showed WT1 mutations in 28 of 211 (13. 2% IntroductionT-lineage acute lymphoblastic leukemia (T-ALL) accounts for 10% to 15% of pediatric and 25% of adult ALL cases. 1 Once associated with a dismal prognosis, the introduction of intensive combination chemotherapy protocols has led to remarkable improvements in survival for this disease. Thus, more than 70% of children and 50% of adult T-ALL patients achieve longlasting complete remissions. 2 However, in contrast with the favorable response to therapy in patients at diagnosis, the prognosis of T-ALL patients with relapsed leukemia remains poor, underscoring the need to identify molecular mechanisms responsible for disease progression and to develop more effective antileukemic drugs active against relapsed T-ALL.WT1 was originally identified as a tumor suppressor gene in patients with the WAGR (Wilms tumor, aniridia, genitourinary abnormalities, mental retardation) tumor predisposition syndrome, caused by inherited germline deletions in the chromosomal band 11p13. 3 Germline mutations in WT1 are also present in patients affected with the Denys-Drash syndrome (DDS), a related tumor predisposition syndrome characterized by pseudohermaphroditism, nephropathy, genital abnormalities, and an increased risk of Wilms tumors. 4 Moreover, biallelic somatic mutations in WT1 account for 15% of sporadic Wilms tumors. 5 In addition to its prominent role in the pathogenesis of Wilms tumors, WT1 mutations have been reported in 10% of cases of acute myeloid leukemia (AML), 20% of biphenotypic leukemias, and sporadic cases of T-ALL. [6][7][8][9] In this study, we aimed to characterize the mechanisms of disease progression and relapse in T-ALL via single nucleotide polymorphism (SNP) array analysis of paired diagnostic and relapsed T-ALL samples. Our results show a remarkable lack of genomic instability in most high-risk T-ALL cases during progression from diagnosis to relapse. In addition, we identified the presence of recurrent chromosomal deletions involving the WT1 locus in T-ALL. Subsequent mutation analysis revealed the presence of WT1 mutations in approximately 10% of pediatric and adult T-ALL samples at diagnosis. WT1 mutations in T-ALL primarily consist of heterozygous frameshift insertions and deletions encoding truncated proteins devoid of the C-termina...

Tumor formation may result from the activation of dominant oncogenes or by inactivation of recessive, tumor suppressor genes. The role of such mutations in the development of pituitary tumors has been studied. Tumors from 88 patients, representing the 4 major classes of adenoma, were investigated. In DNA extracted from matched leukocyte and tumor samples, allelic deletions were sought with 15 probes identifying restriction fragment length polymorphisms on chromosomes 1, 5, 10, 11, 13, 17, 20, and 22. Evidence of amplification or rearrangement of 10 recognized cellular oncogenes (N-ras, mycL1, mycN, myc, H-ras, bcl1, H-stf1, sea, kraS2, and fos) was sought in tumor DNA. Activating dominant mutations of Gs alpha were detected using the polymerase chain reaction to amplify exons 7-10 and hybridizing the product to normal and mutant allele-specific oligonucleotides. Allelic deletions on chromosome 11 were identified in 16 tumors (18%) representing all 4 major subtypes. Deletions on other autosomes were observed in less than 6% of tumors. Three adenomas had deletions on multiple autosomes, 2 of these were aggressive and recurrent. Mutations of Gs alpha were confirmed to be specific to somatotrophinomas, being identified in 36% of such tumors in this series. No evidence of amplification or rearrangement of other recognized cellular oncogenes was found. Inactivation of a recessive oncogene on chromosome 11 is an important and possibly early event in the development of the four major types of pituitary adenoma, whereas activating mutations of Gs alpha are confirmed to be specific to somatotropinomas. Two aggressive tumors were found to have multiple autosomal losses, suggesting a multistep progression in the development of tumors of this phenotype.

Purpose To evaluate the impact of DNMT3A mutations on outcome in younger patients with cytogenetic intermediate-risk acute myeloid leukemia. Patients and Methods Diagnostic samples from 914 patients (97% < 60 years old) were screened for mutations in DNMT3A exons 13 to 23. Clinical outcome was evaluated according to presence or absence of a mutation and stratified according to type of mutation (R882, non-R882 missense, or truncation). Results DNMT3A mutations (DNMT3AMUT) were identified in 272 patients (30%) and associated with a poorer prognosis than wild-type DNMT3A, but the difference was only seen when the results were stratified according to NPM1 genotype. This example of Simpson's paradox results from the high coincidence of DNMT3A and NPM1 mutations (80% of patients with DNMT3AMUT had NPM1 mutations), where the two mutations have opposing prognostic impact. In the stratified analyses, relapse in patients with DNMT3AMUT was higher (hazard ratio, 1.35; 95% CI, 1.07 to 1.72; P = .01), and overall survival was lower (hazard ratio, 1.37; 95% CI, 1.12 to 1.87; P = .002). The impact of DNMT3AMUT did not differ according to NPM1 genotype (test for heterogeneity: relapse, P = .4; overall survival, P = .9). Further analysis according to the type of DNMT3A mutation indicated that outcome was comparable in patients with R882 and non-R882 missense mutants, whereas in those with truncation mutants, it was comparable to wild-type DNMT3A. Conclusion These data confirm that presence of a DNMT3A mutation should be considered as a poor-risk prognostic factor, irrespective of the NPM1 genotype, and suggest that further consideration should be given to the type of DNMT3A mutation.

We investigated the benefit of adding all-trans retinoic acid (ATRA) to chemotherapy for younger patients with nonacute promyelocytic acute myeloid leukemia and high-risk myelodysplastic syndrome, and considered interactions between treatment and molecular markers. Overall, 1075 patients less than 60 years of age were randomized to receive or not receive ATRA in addition to daunorubicin/Ara-C/thioguanine chemotherapy with Ara-C at standard or double standard dose. There were data on FLT3 internal tandem duplications and NPM1 mutations (n ‫؍‬ 592), CEBPA mutations (n ‫؍‬ 423), and MN1 expression (n ‫؍‬ 195). The complete remission rate was 68% with complete remission with incomplete count recovery in an additional 16%; 8-year overall survival was 32%. There was no significant treatment effect for any outcome, with no significant interactions between treatment and demographics, or cytarabine randomization. Importantly, there were no interactions by FLT3/ internal tandem duplications, NPM1, or CEBPA mutation. There was a suggestion that ATRA reduced relapse in patients with lower MN1 levels, but no significant effect on overall survival. Results were consistent when restricted to patients with normal karyotype. ATRA has no overall effect on treatment outcomes in this group of patients. The study did not identify any subgroup of patients likely to derive a significant survival benefit from the addition of ATRA to chemotherapy. This study is registered at http://www. controlled-trials.com under ISRCTN17833622. (Blood. 2010;115:948-956)

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