The Tumor Suppressor Gene Hcdc4 Is Frequently Mutated in Human T-cell Acute Lymphoblastic Leukemia with Functional Consequences for Notch Signaling

Notch signaling is of crucial importance in normal T-cell development and Notch 1 is frequently mutated in T-cell acute lymphoblastic leukemias (TALL), leading to aberrantly high Notch signaling. In this report, we determine whether TALL mutations occur not only in Notch1 but also in the F-box protein hCdc4 (Sel-10, Ago, or Fbxw7), a negative regulator of Notch1. We show that the hCDC4 gene is mutated in leukemic cells from more than 30% of patients with pediatric TALL and derived cell lines. Most hCDC4 mutations found were missense substitutions at critical arginine residues (Arg 465 , Arg 479 , and Arg 505) localized in the substrate-binding region of hCdc4. Cells inactivated for hCdc4 and TALL cells containing hCDC4 mutations exhibited an increased Notch1 protein half-life, consistent with the proposed role of hCdc4 in ubiquitin-dependent proteolysis of Notch1. Furthermore, restoration of wild-type but not mutant hCdc4 in HCT 116 hCDC4-negative cells led to an increased Notch1 ubiquitylation and decreased Notch1 signaling. These results show that hCdc4 mutations interfere with normal Notch1 regulation in vivo. Finally, we found that mutations in hCDC4 and NOTCH1 can occur in the same cancers and that patients carrying hCDC4 and/or NOTCH1 mutations have a favorable overall survival. Collectively , these data show that mutation of hCDC4 is a frequent event in TALL and suggest that hCDC4 mutations and gain-of-function mutations in NOTCH1 might synergize in contributing to the development of pediatric TALL leukemogenesis.


Introduction
T-cell acute lymphocytic leukemia (T-ALL) is an aggressive malignant disease of T-cell precursors, accounting for 10% to 15% of pediatric and 25% of adult ALL cases.The pathogenetic background of T-ALL has mainly been studied in the context of acquired chromosomal translocations resulting in aberrant expression of a number of genes encoding transcription factors important in mammalian development (1).The molecular characterization of a rare t(7;9)(q34;q34.3)translocation that juxtaposes the COOH terminus of the transmembrane receptor Notch1 to the T-cell receptor h (TCRB) locus led to a detailed analysis of Notch1 deregulation in T-ALL (2).This translocation, leading to the expression of a truncated constitutively active form of Notch1 (2), was later shown to be a potent inducer of T-ALL in murine models (2).A more prevalent role for Notch1 in the pathogenesis of T-ALL was established following the discovery of frequent gainof-function mutations in NOTCH1 (1,3).The majority of mutations cluster in regions of the gene encoding the heterodimerization (HD) domain of NOTCH1, but the proline, glutamate, serine, and threonine (PEST) domain also harbors frequent truncating mutations (1)(2)(3).
Notch signaling is tightly regulated by a number of Notchassociated proteins (1) including the human homologue of SEL-10, hCdc4 (also known as Ago and Fbxw7), which has been shown to negatively regulate the intracellular domain (ICD) of Notch1 by mediating its ubiquitin-dependent proteolysis (4).In addition, Cdc4 knockout mice die in utero at embryonic day 10.5, manifesting elevated Notch ICD levels and marked abnormalities in vascular development (5,6).
hCdc4 is a member of the F-box family of proteins, which are interchangeable substrate recognition subunits of SCF ubiquitin ligases (7).In the case of hCdc4, substrate recognition is mediated through interaction with eight adjacent WD40 protein binding motifs located in the COOH terminus (8).hCdc4 has also been shown to mediate the ubiquitin-dependent proteolysis of several other proteins important in regulating cellular division including cyclin E (9, 10), c-Myc, c-Jun, and Aurora-A (7).The role of hCdc4 as a negative regulator of several oncoproteins has suggested that it may function as a tumor suppressor in human malignancies.In support of this, mutations of hCDC4 have been detected in several human tumor types (11,12), and inactivation of hCdc4 has been shown to lead to an increased genetic instability (11).Interestingly, although Cdc4 heterozygous mice do not develop spontaneous tumors (5,6), heterozygous loss of both Cdc4 and p53 causes predisposition to development of a variety of epithelial tumors in mice (13).
In this report, we show that hCDC4 is frequently mutated in leukemic cells from children with T-ALL and that these mutations are associated with decreased Notch1 ubiquitylation and increased Notch1 protein stability.Finally, we find that patients with hCDC4 and/or NOTCH1 mutations have a favorable outcome compared with patients without alterations in these two components.

Materials and Methods
Patients and cell lines.Samples of cryopreserved lymphoblasts from 26 children with T-ALL and 20 patients with ALL of B-precursor phenotype, treated according to high-risk protocols prepared by the Nordic Society of Pediatric Hematology and Oncology (NOPHO; see Table 2 1).The patients were treated according to NOPHO-ALL 86 for patients diagnosed 1988-1991 (n = 4), NOPHO ALL-92 for patients diagnosed 1992-2000 (n = 14), and NOPHO ALL-2000 for patients diagnosed from 2001 onwards (n = 8; ref. 14).Normal cells were obtained from peripheral blood or bone marrow following remission induced by chemotherapy.Leukemic and normal cells were purified as previously described (15).Approval was obtained from the Institutional Review Board for these studies.Malignant T-ALL and pre-B-ALL and other cell lines (Table 1) were cultured as described (9,11,16).
Mutation detection.Genomic DNA was prepared from leukemic and normal cells and derived cell lines using a commercial kit according to the manufacturer's instructions (Qiagen).Mutation analysis of NOTCH1 and hCDC4 was done as previously described (3,12).PCR primers and singlestrand conformational polymorphism (SSCP) conditions are available on request.
Notch1 stability and activity analysis.Preparation of protein extracts, electrophoresis, and Western blotting have been described (9,12).For protein stability experiments, cells were treated with 100 Ag/mL cycloheximide (Biomol) and 5 mmol/L EDTA for 15 min where indicated . NOTCH1 and hCDC4 expression constructs (4,18), transient short interfering RNA (siRNA) transfections, hCDC4 expression analysis, and NOTCH1 gene reporter assays have been described (4,18).In vitro mutation of hCdc4 Arg 465 , Arg 479 , and Arg 505 was conducted using the in vitro mutagenesis QuickChange kit (Strategene).Antibodies to MYC (anti-9E10, Santa Cruz Biotechnology), hemagglutinin (anti-HA, Santa Cruz Biotechnology), and the cleaved, activated (ICD) form of Notch 1 (Cell Signaling) were used as recommended by the manufacturers.Tubulin antibodies (Sigma) were used as a control for loading.Horseradish peroxidase-conjugated antirabbit and antimouse (Cell Signaling) antibodies were used as secondary antibodies.Ectopically expressed hCdc4 was detected with a rabbit polyclonal antibody raised against the WD40 domains of hCdc4 (9).
Statistics.The significance of correlations between hCDC4 mutations, NOTCH1 mutation, and clinical variables was determined using Fisher's exact test (Table 2).The Kaplan-Meier method was used to generate graphs and estimate event-free survival.Differences in the prognosis between groups were evaluated using the log-rank test.

Results and Discussion
PCR-SSCP analysis was used to screen the entire coding region of hCDC4 in pediatric leukemic T-and B-lineage ALLs (12).No band shifts were observed in 20 B-lineage ALL leukemia samples analyzed (data not shown).However, several aberrantly migrating bands, indicative of mutations, were detected in 8 of 26 T-ALL specimens (Fig. 1A and data not shown).Sequence analysis showed that the specimens contained mutations that resulted in missense substitutions at codons Arg 465 (three patients; exon 8), Arg 479 ( four patients; exon 9), or Arg 689 (one patient; exon 11) in hCdc4 (Fig. 1A; Table 1).Codons Arg 465 and Arg 479 are localized to the surface of the h-propeller structure of hCdc4 (8), which is essential for recognition of phosphorylated cyclin E1 in mammalian cells (9)(10)(11) and the cyclin-dependent kinase inhibitor Sic1 in yeast (8).Arg 689 is localized in the eighth h-propeller blade of hCdc4 (8) and is conserved in human, mouse, worm, and flies.This particular *P values from the Fisher exact test.Because multiple comparisons were made, a Bonferroni correction was made for the analysis of presenting clinical features associated with genotype ( five analyses).Two other analyses, the association between outcome (survival) and Notch1/hCDC4 genotype and CDKN2A status and Notch1/hCDC4 genotype, were considered as distinct and were thus not corrected for.Statistically significant test results (P V 0.01 for the association with presenting clinical features and P V 0.05 for the other analyses) are given in boldface.cLymphomatous features defined in this population as T-cell disease + one of mediastinal mass, splenomegaly below the navel, and lymphadenopathy with nodes >3 cm in size.bEvent defined as resistant disease (no remission after induction course, salvage therapy initiated; n = 2, both wild-type for NOTCH1 and hCDC4); induction death (dead during induction; n = 0); relapse (n = 5, one of which was hCDC4 mutant, but NOTCH1 wild-type, the remaining 4 were wild-type for both genes); dead in CR1 (n = 0); or second malignant neoplasm (n = 0).
x Patients not analyzed for CDKN2A status excluded from the analysis.Research.
on April 9, 2017.© 2007 American Association for Cancer cancerres.aacrjournals.orgDownloaded from mutation has not been previously described in human cancers and it remains to be shown if it affects substrate binding or if it may destabilize the blades of the propeller in hCdc4.Importantly, hCDC4 mutations were absent in cells from matching nonmalignant leukocytes obtained from the same patients, indicating that the mutations were of somatic origin (data not shown).Together, these data show that >30% of patients with pediatric T-ALL harbor hCDC4 mutations, the highest mutation frequency reported in the hCDC4 gene in any human tumor type to date.
Additionally, we did mutational analysis of hCDC4 in a panel of cell lines derived from various hematologic malignancies, particularly of T-cell origin (Table 1; ref. 16).Nine malignant T-cell lines were screened for mutations, and in four of those, Jurkat, JM, CCRF-CEM, and P12-ISHIKAWA, hCDC4 was mutated (Table 1).Three T-cell lines (Jurkat, JM, and P12) had missense mutations that changed Arg 505 of hCdc4, whereas CCRF-CEM had a mutation at Arg 465 (Table 1), as previously reported (10).Thus, similar amino acid changes at critical arginine residues in the binding pocket of hCdc4 occur in both primary leukemic T cells and T-cell lines (Table 1).No hCDC4 mutations were identified in any of the other leukemia or lymphoma cell lines, with the exception of the multiple myeloma cell line U266-1984, which harbored an Arg 505 missense mutation (Table 1).
Western blot analysis of lysates prepared from T-ALL cell lines with hCDC4 mutations indicated that these cells had elevated Notch1 ICD protein levels, as compared with T-ALL cell lines Figure 1.hCDC4 mutations associate with decreased ubiquitylation and increased Notch1 ICD stability.All 13 exons of hCDC4 were amplified and mutations identified using SSCP analysis as described (12).Each mutation was confirmed by direct sequence analysis using genomic tumor DNA as a template.Matched normal DNA from each tumor was amplified and sequenced as control.A, SSCP analysis of hCDC4 exon 8 in three T-ALL specimens.Left, an aberrantly migrating band is evident in lane 2 and sequence analysis of this tumor sample reveals a G to A substitution, causing arginine to change to histidine at codon 465 in hCdc4 (right ).Middle, sequence chromatogram of matched normal DNA from the same patient.B, cycloheximide-chase experiments assessing Notch1 ICD stability in different cell lines.Notch1 ICD was first induced by treating cells with EDTA (EDTA induces cleavage and release of membrane-bound Notch1; ref. 17) and subsequently chasing with 100 Ag/mL cycloheximide for the indicated times.Tubulin protein level is shown as a control for loading.Quantitation was done using a charge-coupled device camera and an image analyzer (Image Reader LAS-1000, Fuji film).C, Notch1 ICD levels in hCDC4 -silenced MCF-7 cells.hCDC4 was first silenced by means of siRNA transfection for 48 h, treatment with EDTA for 15 min, and then chasing with cycloheximide for 2 h.Tubulin protein level is shown as a control for loading.D, in vivo ubiquitylation of Notch1 ICD in HCT 116 hCDC4 -negative cells.Cells were cotransfected with MYC-tagged Notch1 ICD, hemagglutinin-tagged ubiquitin, and either glutathione S-transferase (GST )-tagged wt hCdc4 or different GST-tagged arginine mutant versions of hCdc4 expression plasmids.Before harvesting, cells were treated with 30 Amol/L MG-132 to preserve multiubiquitin chains of Notch1 ICD, and Notch1 ICD was subsequently precipitated with myc-agarose beads.Multiubiquitylated Notch1 ICD conjugates were detected by Western blot analysis with hemagglutinin antibodies as previously described (18).
without mutation in either Notch1 or hCdc4 (Supplementary Fig. S1A).To specifically address whether elevated level of Notch1 is due to reduced degradation of the Notch1 ICD, we did cycloheximide-chase experiments in cell lines with specific genetic alterations in hCDC4 and NOTCH1.Indeed, cell lines with either NOTCH1 and/or hCDC4 mutations exhibited a reduced rate of Notch1 ICD degradation compared with cell lines without alterations in these genes (Fig. 1B; Table 1).These results were also recapitulated in T-ALL cells treated with the g-secretase inhibitor DAPT to avoid potential differences in the rate of ICD production among these cell lines (Supplementary Fig. S1B).We next analyzed Notch1 ICD degradation in human embryonic kidney HEK 293, breast cancer MCF-7, and colon carcinoma HCT 116 cells following Notch1 activation by EDTA treatment.Notch1 ICD was rapidly degraded in all cell lines, consistent with the wildtype NOTCH1 and hCDC4 genotypes in these cells (Fig. 1B and data not shown).In contrast, HCT 116 hCDC4-negative cells (11) exhibited a clear stabilization of Notch1 ICD compared with the parental HCT 116 cells (Fig. 1B).Notably, silencing of hCDC4 using siRNA duplexes also significantly reduced the rate of turnover of Notch1 ICD in both MCF-7 and HEK 293 cells (Fig. 1C and data not shown), consistent with the proposed role of hCdc4 in degradation of Notch1 ICD.As expected, Notch1 ICD was rapidly degraded in pre-B-ALL cells that do not carry hCDC4 and/or NOTCH1 mutations (data not shown).Finally, to directly address whether the specific hCdc4 arginine mutants identified in T-ALL tumors affect the formation of multiubiquitin chains on Notch1 ICD, we did in vivo ubiquitylation assays in hCdc4-negative cells reconstituted with either a wild-type hCdc4 allele or with Arg 465 and Arg 505 mutant versions of hCdc4.As shown in Fig. 1D, each of the mutant alleles was severely deficient in multiubiquitylating Notch1 ICD as compared with wild-type hCdc4.
Given the presence of mutations in both NOTCH1 and hCDC4 in T-ALL cell lines (Table 1), we next explored whether both genes are simultaneously mutated also in primary T-ALL samples.As can be seen in Table 1, leukemic samples from eight patients contain NOTCH1 mutations in the HD domain alone ( four patients), the PEST domain alone (one patient), or both domains (three patients).Two patient samples with NOTCH1 mutations were also found to contain hCDC4 mutations (Table 1).In concordance with previous studies (3), mutations in NOTCH1 were not observed in any of the B-ALL cases (data not shown).A total of 11 NOTCH1 mutations were detected in 8 of 26 T-ALL specimens (Table 1), a mutation frequency comparable with that previously described (19,20).The NOTCH1 mutations identified in this study were similar to previously reported (3,20) ''hotspots'' within the HD domain and truncating insertions and deletions in the PEST domain (Table 1).Interestingly, one tumor (patient #26) was found to harbor a NOTCH1 missense mutation that results in a Thr to Met substitution at position 2,484 in the PEST domain (Table 1), and point mutations in the PEST domain that are not frameshift or nonsense mutations are known to be infrequent in T-ALL (3,20).Several hCdc4 target substrates, including cyclin E1 and c-Myc, contain a so-called hCdc4 phosphodegron motif (CPD), which is essential for interaction with SCF hCdc4 and their subsequent ubiquitylation (8).Of note, the amino acids surrounding Thr 2484 of Notch1 resemble a consensus CPD motif, suggesting that the T2484M PEST mutation might prevent its recognition by hCdc4.However, several truncating PEST domain mutations occur downstream of this motif (Table 1), suggesting that multiple CPD motifs may be present in Notch1 ICD and regulate its degradation (21).
Because one function of hCdc4 is the regulation of ubiquitylation and degradation of Notch1 ICD, we also wanted to examine the influence of the hCdc4 arginine mutants on Notch1 signaling.Introduction of a synthetic Notch1-sensitive reporter construct into HCT 116 hCDC4-negative cells, together with a wild-type hCdc4 construct, caused a significant reduction in Notch1 activity (Fig. 2A;

Figure 2 .
Figure 2. Effects of hCDC4 mutations on Notch1 signaling and patient outcome.HCT 116 hCDC4 -negative cells (A) and U2OS cells (B ) were transiently transfected with Notch1 DE (200 ng), wild-type or specific hCdc4 arginine mutants (1 Ag), 12XCSL-luciferase construct (1 Ag), and a Renilla luciferase internal control plasmid.Luciferase activity was measured using the Dual-Luciferase Reporter Assay System according to the manufacturer's instructions (Promega).C, patients with hCDC4/NOTCH1 mutations have a favorable overall survival.Kaplan-Meier estimate of event-free survival (EFS ) at 10 y in pediatric T-ALL patients.Patients were classified according to the presence or absence of hCDC4/NOTCH1 mutations.