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1.
JCO Precis Oncol ; 7: e2300302, 2023 Sep.
Article in English | MEDLINE | ID: mdl-37944074

ABSTRACT

PURPOSE: Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric malignancy with myelodysplastic and myeloproliferative features. Curative treatment is restricted to hematopoietic stem-cell transplantation. Fludarabine combined with cytarabine (FLA) and 5-azacitidine (AZA) monotherapy are commonly used pre-transplant therapies. Here, we present a drug screening strategy using a flow cytometry-based precision medicine platform to identify potential additional therapeutic vulnerabilities. METHODS: We screened 120 dual- and 10 triple-drug combinations (DCs) on peripheral blood (n = 21) or bone marrow (n = 6) samples from 27 children with JMML to identify DCs more effectively reducing leukemic cells than the DCs' components on their own. If fewer leukemic cells survived a DC ex vivo treatment compared with that DC's most effective component alone, the drug effect was referred to as cooperative. The difference between the two resistant fractions is the effect size. RESULTS: We identified 26 dual- and one triple-DC more effective than their components. The differentiation agent tretinoin (TRET; all-trans retinoic acid) reduced the resistant fraction of FLA in 19/21 (90%) samples (decrease from 15% [2%-61%] to 11% [2%-50%] with a mean effect size of 3.8% [0.5%-11%]), and of AZA in 19/25 (76%) samples (decrease from 69% [34%-100+%] to 47% [17%-83%] with a mean effect size of 16% [0.3%-40%]). Among the resistant fractions, the mean proportion of CD38+ cells increased from 7% (0.03%-25%; FLA) to 17% (0.3%-38%; FLA + TRET) or from 10% (0.2%-31%; AZA) to 51% (0.8%-88%; AZA + TRET). CONCLUSION: TRET enhanced the effects of FLA and AZA in ex vivo assays with primary JMML samples.


Subject(s)
Hematopoietic Stem Cell Transplantation , Leukemia, Myelomonocytic, Juvenile , Child , Humans , Leukemia, Myelomonocytic, Juvenile/drug therapy , Leukemia, Myelomonocytic, Juvenile/diagnosis , Leukemia, Myelomonocytic, Juvenile/pathology , Tretinoin/pharmacology , Tretinoin/therapeutic use , Azacitidine/therapeutic use
2.
Cancers (Basel) ; 14(24)2022 Dec 18.
Article in English | MEDLINE | ID: mdl-36551725

ABSTRACT

Acute myeloid leukemia (AML) is a heterogeneous disease that accounts for ~20% of all childhood leukemias, and more than 40% of children with AML relapse within three years of diagnosis. Although recent efforts have focused on developing a precise medicine-based approach towards treating AML in adults, there remains a critical gap in therapies designed specifically for children. Here, we present ex vivo drug sensitivity profiles for children with de novo AML using an automated flow cytometry platform. Fresh diagnostic blood or bone marrow aspirate samples were screened for sensitivity in response to 78 dose conditions by measuring the reduction in leukemic blasts relative to the control. In pediatric patients treated with conventional chemotherapy, comprising cytarabine, daunorubicin and etoposide (ADE), ex vivo drug sensitivity results correlated with minimal residual disease (r = 0.63) and one year relapse-free survival (r = 0.70; AUROC = 0.94). In the de novo ADE analysis cohort of 13 patients, AML cells showed greater sensitivity to bortezomib/panobinostat compared with ADE, and comparable sensitivity between venetoclax/azacitidine and ADE ex vivo. Two patients showed a differential response between ADE and bortezomib/panobinostat, thus supporting the incorporation of ex vivo drug sensitivity testing in clinical trials to further evaluate the predictive utility of this platform in children with AML.

3.
Front Oncol ; 12: 832816, 2022.
Article in English | MEDLINE | ID: mdl-35223511

ABSTRACT

Blocking the pyrimidine nucleotide de novo synthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH) results in the cell cycle arrest and/or differentiation of rapidly proliferating cells including activated lymphocytes, cancer cells, or virally infected cells. Emvododstat (PTC299) is an orally bioavailable small molecule that inhibits DHODH. We evaluated the potential for emvododstat to inhibit the progression of acute myeloid leukemia (AML) using several in vitro and in vivo models of the disease. Broad potent activity was demonstrated against multiple AML cell lines, AML blasts cultured ex vivo from patient blood samples, and AML tumor models including patient-derived xenograft models. Emvododstat induced differentiation, cytotoxicity, or both in primary AML patient blasts cultured ex vivo with 8 of 10 samples showing sensitivity. AML cells with diverse driver mutations were sensitive, suggesting the potential of emvododstat for broad therapeutic application. AML cell lines that are not sensitive to emvododstat are likely to be more reliant on the salvage pathway than on de novo synthesis of pyrimidine nucleotides. Pharmacokinetic experiments in rhesus monkeys demonstrated that emvododstat levels rose rapidly after oral administration, peaking about 2 hours post-dosing. This was associated with an increase in the levels of dihydroorotate (DHO), the substrate for DHODH, within 2 hours of dosing indicating that DHODH inhibition is rapid. DHO levels declined as drug levels declined, consistent with the reversibility of DHODH inhibition by emvododstat. These preclinical findings provide a rationale for clinical evaluation of emvododstat in an ongoing Phase 1 study of patients with relapsed/refractory acute leukemias.

4.
Blood ; 137(6): 751-762, 2021 02 11.
Article in English | MEDLINE | ID: mdl-32929488

ABSTRACT

Approximately 50% of acute myeloid leukemia (AML) patients do not respond to induction therapy (primary induction failure [PIF]) or relapse after <6 months (early relapse [ER]). We have recently shown an association between an immune-infiltrated tumor microenvironment (TME) and resistance to cytarabine-based chemotherapy but responsiveness to flotetuzumab, a bispecific DART antibody-based molecule to CD3ε and CD123. This paper reports the results of a multicenter, open-label, phase 1/2 study of flotetuzumab in 88 adults with relapsed/refractory AML: 42 in a dose-finding segment and 46 at the recommended phase 2 dose (RP2D) of 500 ng/kg per day. The most frequent adverse events were infusion-related reactions (IRRs)/cytokine release syndrome (CRS), largely grade 1-2. Stepwise dosing during week 1, pretreatment dexamethasone, prompt use of tocilizumab, and temporary dose reductions/interruptions successfully prevented severe IRR/CRS. Clinical benefit accrued to PIF/ER patients showing an immune-infiltrated TME. Among 30 PIF/ER patients treated at the RP2D, the complete remission (CR)/CR with partial hematological recovery (CRh) rate was 26.7%, with an overall response rate (CR/CRh/CR with incomplete hematological recovery) of 30.0%. In PIF/ER patients who achieved CR/CRh, median overall survival was 10.2 months (range, 1.87-27.27), with 6- and 12-month survival rates of 75% (95% confidence interval [CI], 0.450-1.05) and 50% (95% CI, 0.154-0.846). Bone marrow transcriptomic analysis showed that a parsimonious 10-gene signature predicted CRs to flotetuzumab (area under the receiver operating characteristic curve = 0.904 vs 0.672 for the European LeukemiaNet classifier). Flotetuzumab represents an innovative experimental approach associated with acceptable safety and encouraging evidence of activity in PIF/ER patients. This trial was registered at www.clinicaltrials.gov as #NCT02152956.


Subject(s)
Antineoplastic Agents, Immunological/therapeutic use , Immunotherapy , Leukemia, Myeloid, Acute/therapy , Salvage Therapy , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cytokine Release Syndrome/chemically induced , Cytokine Release Syndrome/drug therapy , Dose-Response Relationship, Immunologic , Drug Administration Schedule , Drug Resistance, Neoplasm , Female , Follow-Up Studies , Hematopoiesis/drug effects , Humans , Leukemia, Myeloid, Acute/drug therapy , Male , Maximum Tolerated Dose , Middle Aged , Nausea/chemically induced , Protein Interaction Maps , Survival Rate
5.
Blood Adv ; 4(12): 2768-2778, 2020 06 23.
Article in English | MEDLINE | ID: mdl-32569379

ABSTRACT

Precision medicine approaches such as ex vivo drug sensitivity screening (DSS) are appealing to inform rational drug selection in myelodysplastic syndromes (MDSs) and acute myeloid leukemia, given their marked biologic heterogeneity. We evaluated a novel, fully automated ex vivo DSS platform that uses high-throughput flow cytometry in 54 patients with newly diagnosed or treatment-refractory myeloid neoplasms to evaluate sensitivity (blast cytotoxicity and differentiation) to 74 US Food and Drug Administration-approved or investigational drugs and 36 drug combinations. After piloting the platform in 33 patients, we conducted a prospective feasibility study enrolling 21 patients refractory to hypomethylating agents (HMAs) to determine whether this assay could be performed within a clinically actionable time frame and could accurately predict clinical responses in vivo. When assayed for cytotoxicity, ex vivo drug sensitivity patterns were heterogeneous, but they defined distinct patient clusters with differential sensitivity to HMAs, anthracyclines, histone deacetylase inhibitors, and kinase inhibitors (P < .001 among clusters) and demonstrated synergy between HMAs and venetoclax (P < .01 for combinations vs single agents). In our feasibility study, ex vivo DSS results were available at a median of 15 days after bone marrow biopsy, and they informed personalized therapy, which frequently included venetoclax combinations, kinase inhibitors, differentiative agents, and androgens. In 21 patients with available ex vivo and in vivo clinical response data, the DSS platform had a positive predictive value of 0.92, negative predictive value of 0.82, and overall accuracy of 0.85. These data demonstrate the utility of this approach for identifying potentially useful and often novel therapeutic drugs for patients with myeloid neoplasms refractory to standard therapies.


Subject(s)
Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Pharmaceutical Preparations , Drug Evaluation, Preclinical , Humans , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/drug therapy , Myelodysplastic Syndromes/diagnosis , Myelodysplastic Syndromes/drug therapy , Prospective Studies , United States
6.
Clin Cancer Res ; 26(3): 726-737, 2020 02 01.
Article in English | MEDLINE | ID: mdl-31719049

ABSTRACT

PURPOSE: A cryptic inv(16)(p13.3q24.3) encoding the CBFA2T3-GLIS2 fusion is associated with poor outcome in infants with acute megakaryocytic leukemia. We aimed to broaden our understanding of the pathogenesis of this fusion through transcriptome profiling. EXPERIMENTAL DESIGN: Available RNA from children and young adults with de novo acute myeloid leukemia (AML; N = 1,049) underwent transcriptome sequencing (mRNA and miRNA). Transcriptome profiles for those with the CBFA2T3-GLIS2 fusion (N = 24) and without (N = 1,025) were contrasted to define fusion-specific miRNAs, genes, and pathways. Clinical annotations defined distinct fusion-associated disease characteristics and outcomes. RESULTS: The CBFA2T3-GLIS2 fusion was restricted to infants <3 years old (P < 0.001), and the presence of this fusion was highly associated with adverse outcome (P < 0.001) across all morphologic classifications. Further, there was a striking paucity of recurrent cooperating mutations, and transduction of cord blood stem cells with this fusion was sufficient for malignant transformation. CBFA2T3-GLIS2 positive cases displayed marked upregulation of genes with cell membrane/extracellular matrix localization potential, including NCAM1 and GABRE. Additionally, miRNA profiling revealed significant overexpression of mature miR-224 and miR-452, which are intronic miRNAs transcribed from the GABRE locus. Gene-set enrichment identified dysregulated Hippo, TGFß, and hedgehog signaling, as well as NCAM1 (CD56) interaction pathways. Therapeutic targeting of fusion-positive leukemic cells with CD56-directed antibody-drug conjugate caused significant cytotoxicity in leukemic blasts. CONCLUSIONS: The CBFA2T3-GLIS2 fusion defines a highly refractory entity limited to infants that appears to be sufficient for malignant transformation. Transcriptome profiling elucidated several highly targetable genes and pathways, including the identification of CD56, providing a highly plausible target for therapeutic intervention.


Subject(s)
Biomarkers, Tumor/genetics , Gene Expression Profiling , Leukemia, Myeloid, Acute/genetics , MicroRNAs/genetics , Mutation , Oncogene Proteins, Fusion/genetics , Adult , CD56 Antigen/genetics , Child, Preschool , Female , Follow-Up Studies , Humans , Infant , Infant, Newborn , Leukemia, Myeloid, Acute/pathology , Male , Middle Aged , Prognosis , RNA, Messenger , Receptors, GABA-A/genetics , Young Adult
7.
Clin Cancer Res ; 21(18): 4165-73, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26015513

ABSTRACT

PURPOSE: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival. EXPERIMENTAL DESIGN: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody-drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification. RESULTS: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification. CONCLUSIONS: These findings demonstrate the potential of PF-06647263 (anti-EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development.


Subject(s)
Aminoglycosides/chemistry , Antibodies, Monoclonal, Murine-Derived/chemistry , Enediynes/chemistry , Ephrin-A4/chemistry , Ovarian Neoplasms/drug therapy , Triple Negative Breast Neoplasms/drug therapy , Animals , Antibodies, Monoclonal, Humanized/chemistry , Antigens, Neoplasm/chemistry , Cell Line, Tumor , DNA/chemistry , Drug Design , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Neoplastic Stem Cells/metabolism , Prospective Studies , Random Allocation , Treatment Outcome , Xenograft Model Antitumor Assays
8.
Lab Invest ; 93(9): 970-82, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23917877

ABSTRACT

Cancer is a heterogeneous disease manifest in many forms. Tumor histopathology can differ significantly among patients and cellular heterogeneity within tumors is common. A primary goal of cancer biologists is to better understand tumorigenesis and cancer progression; however, the complex nature of tumors has posed a substantial challenge to unlocking cancer's secrets. The cancer stem cell (CSC) paradigm for the pathobiology of solid tumors appropriately acknowledges phenotypic and functional tumor cell heterogeneity observed in solid tumors and accounts for the disconnect between drug approval based on response and the general inability of approved therapies to meaningfully impact survival due to their failure to eradicate these most important of cellular targets. First proposed to exist decades ago, CSC have only recently begun to be precisely identified due to technical advancements that facilitate identification, isolation, and interrogation of distinct tumor cell subpopulations with differing ability to form and perpetuate tumors. Precise identification of CSC populations and the complete hierarchy of cells within solid tumors will facilitate more accurate characterization of patient subtypes and ultimately contribute to more personalized and effective therapies. Rapid advancement in the understanding of tumor biology as it exists in patients requires cooperation among institutions, surgeons, pathologists, cancer biologists and patients alike, primarily because this translational research is best done with patient-derived tissue grown in the xenograft setting as patient-derived xenografts. This review calls for a broader change in the approaches taken to study cancer pathobiology, highlights what implications the CSC paradigm has for pathologists and cancer biologists alike, and calls for greater collaboration between institutions, physicians and scientists in order to more rapidly advance our collective understanding of cancer.


Subject(s)
Neoplasm Transplantation/methods , Neoplasms/pathology , Neoplastic Stem Cells/pathology , Transplantation, Heterologous/pathology , Animals , Humans , Medical Oncology
9.
J Biol Chem ; 285(43): 32834-32843, 2010 Oct 22.
Article in English | MEDLINE | ID: mdl-20729212

ABSTRACT

The p110 CUX1 homeodomain protein participates in the activation of DNA replication genes in part by increasing the affinity of E2F factors for the promoters of these genes. CUX1 expression is very weak in quiescent cells and increases during G(1). Biochemical activities associated with transcriptional activation by CUX1 are potentiated by post-translational modifications in late G(1), notably a proteolytic processing event that generates p110 CUX1. Constitutive expression of p110 CUX1, as observed in some transformed cells, leads to accelerated entry into the S phase. In this study, we investigated the post-translation regulation of CUX1 during mitosis and the early G(1) phases of proliferating cells. We observed a major electrophoretic mobility shift and a complete inhibition of DNA binding during mitosis. We show that cyclin B/CDK1 interacts with CUX1 and phosphorylates it at multiple sites. Serine to alanine replacement mutations at 10 SP dipeptide sites were required to restore DNA binding in mitosis. Passage into G(1) was associated with the degradation of some p110 CUX1 proteins, and the remaining proteins were gradually dephosphorylated. Indirect immunofluorescence and subfractionation assays using a phospho-specific antibody showed that most of the phosphorylated protein remained in the cytoplasm, whereas the dephosphorylated protein was preferentially located in the nucleus. Globally, our results indicate that the hyperphosphorylation of CUX1 by cyclin B/CDK1 inhibits its DNA binding activity in mitosis and interferes with its nuclear localization following cell division and formation of the nuclear membrane, whereas dephosphorylation and de novo synthesis contribute to gradually restore CUX1 expression and activity in G(1).


Subject(s)
CDC2 Protein Kinase/metabolism , Cell Nucleus/metabolism , Cyclin B/metabolism , G1 Phase/physiology , Homeodomain Proteins/metabolism , Mitosis/physiology , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Active Transport, Cell Nucleus/physiology , Animals , CDC2 Protein Kinase/genetics , Cell Line , Cell Nucleus/genetics , Cyclin B/genetics , DNA/genetics , DNA/metabolism , Homeodomain Proteins/genetics , Humans , Mice , Mutation, Missense , Nuclear Proteins/genetics , Phosphorylation/physiology , Protein Binding/physiology , Repressor Proteins/genetics
10.
Cancer Cell ; 15(4): 341-52, 2009 Apr 07.
Article in English | MEDLINE | ID: mdl-19345332

ABSTRACT

Loss of the JunB/AP-1 transcription factor induces a myeloproliferative disease (MPD) arising from the hematopoietic stem cell (HSC) compartment. Here, we show that junB inactivation deregulates the cell-cycle machinery and increases the proliferation of long-term repopulating HSCs (LT-HSCs) without impairing their self-renewal or regenerative potential in vivo. We found that JunB loss destabilizes a complex network of genes and pathways that normally limit myeloid differentiation, leading to impaired responsiveness to both Notch and TGF-beta signaling due in part to transcriptional deregulation of the Hes1 gene. These results demonstrate that LT-HSC proliferation and differentiation are uncoupled from self-renewal and establish some of the mechanisms by which JunB normally limits the production of myeloid progenitors, hence preventing initiation of myeloid malignancies.


Subject(s)
Cell Differentiation , Cell Proliferation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Myeloproliferative Disorders/metabolism , Proto-Oncogene Proteins c-jun/physiology , Animals , Cell Cycle , Cells, Cultured , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloproliferative Disorders/prevention & control , Receptors, Notch/metabolism , Signal Transduction , Transforming Growth Factor beta/metabolism
11.
PLoS Biol ; 7(3): e59, 2009 Mar 17.
Article in English | MEDLINE | ID: mdl-19296721

ABSTRACT

How oncogenes modulate the self-renewal properties of cancer-initiating cells is incompletely understood. Activating KRAS and NRAS mutations are among the most common oncogenic lesions detected in human cancer, and occur in myeloproliferative disorders (MPDs) and leukemias. We investigated the effects of expressing oncogenic Kras(G12D) from its endogenous locus on the proliferation and tumor-initiating properties of murine hematopoietic stem and progenitor cells. MPD could be initiated by Kras(G12D) expression in a highly restricted population enriched for hematopoietic stem cells (HSCs), but not in common myeloid progenitors. Kras(G12D) HSCs demonstrated a marked in vivo competitive advantage over wild-type cells. Kras(G12D) expression also increased the fraction of proliferating HSCs and reduced the overall size of this compartment. Transplanted Kras(G12D) HSCs efficiently initiated acute T-lineage leukemia/lymphoma, which was associated with secondary Notch1 mutations in thymocytes. We conclude that MPD-initiating activity is restricted to the HSC compartment in Kras(G12D) mice, and that distinct self-renewing populations with cooperating mutations emerge during cancer progression.


Subject(s)
Hematopoietic Stem Cells/pathology , Leukemia, Experimental/pathology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins p21(ras)/metabolism , Animals , Base Sequence , Cell Proliferation , Cell Transformation, Neoplastic , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Leukemia, Experimental/metabolism , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Myeloproliferative Disorders/metabolism , Myeloproliferative Disorders/pathology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Stem Cells/metabolism , Stem Cells/pathology
12.
J Biol Chem ; 280(38): 32712-21, 2005 Sep 23.
Article in English | MEDLINE | ID: mdl-16081423

ABSTRACT

Previous experiments with peptide fusion proteins suggested that cyclin A/Cdk1 and Cdk2 might exhibit similar yet distinct phosphorylation specificities. Using a physiological substrate, CDP/Cux, our study confirms this notion. Proteolytic processing of CDP/Cux by cathepsin L generates the CDP/Cux p110 isoform at the beginning of S phase. CDP/Cux p110 makes stable interactions with DNA during S phase but is inhibited in G2 following the phosphorylation of serine 1237 by cyclin A/Cdk1. In this study, we propose that differential phosphorylation by cyclin A/Cdk1 and cyclin A/Cdk2 enables CDP/Cux p110 to exert its function as a transcriptional regulator specifically during S phase. We found that like cyclin A/Cdk1, cyclin A/Cdk2 interacted efficiently with recombinant CDP/Cux proteins that contain the Cut homeodomain and an adjacent cyclin-binding motif (Cy). In contrast to cyclin A/Cdk1, however, cyclin A/Cdk2 did not efficiently phosphorylate CDP/Cux p110 on serine 1237 and did not inhibit its DNA binding activity in vitro. Accordingly, co-expression with cyclin A/Cdk2 in cells did not inhibit the DNA binding and transcriptional activities of CDP/Cux p110. To confirm that the sequence surrounding serine 1237 was responsible for the differential regulation by Cdk1 and Cdk2, we replaced 4 amino acids flanking the phosphorylation site to mimic a known Cdk2 phosphorylation site present in the Cdc6 protein. Both cyclin A/Cdk2 and Cdk1 efficiently phosphorylated the CDP/Cux(Cdc6) mutant and inhibited its DNA binding activity. Altogether our results help explain why the DNA binding activity of CDP/Cux p110 is maximal during S phase and decreases in G2 phase.


Subject(s)
CDC2 Protein Kinase/metabolism , Cyclin A/metabolism , Gene Expression Regulation , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Amino Acid Motifs , Animals , Binding Sites , Cathepsin L , Cathepsins/metabolism , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Nucleus/metabolism , Cysteine Endopeptidases/metabolism , DNA/chemistry , DNA/metabolism , G2 Phase , Glutathione Transferase/metabolism , Homeodomain Proteins , Immunoblotting , Luciferases/metabolism , Mice , Models, Genetic , NIH 3T3 Cells , Oligonucleotides/chemistry , Phosphorylation , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Proteins/chemistry , S Phase , Saccharomyces cerevisiae Proteins/metabolism , Serine/chemistry , Transcription, Genetic
13.
Int J Cancer ; 100(4): 429-32, 2002 Aug 01.
Article in English | MEDLINE | ID: mdl-12115525

ABSTRACT

Genetic analyses and mRNA expression studies have implicated CUTL1 as a candidate tumor-suppressor gene in uterine leiomyomas and breast cancers. However, modulation of CDP/Cux, the protein encoded by CUTL1, does not agree with this notion. The activity of CDP/Cux, which is the DNA binding subunit of HiNF-D, was upregulated as normal cells progressed into S phase and constitutively elevated in several tumor cell lines. Activation of CDP/Cux at the G(1)/S transition involved the proteolytic processing of the protein to generate a shorter isoform. Uterine leiomyomas represent a unique reagent for molecular analysis because they are resected as homogeneous tumor tissue together with the adjacent normal myometrium and they are often very large. In the present study, proteins were isolated from 16 pairs of matched tumors and adjacent myometrium and analyzed by Western blot and electrophoretic mobility shift assays. Strikingly, in 11/16 tumors, the steady-state level of small CDP/Cux isoforms was increased compared to normal control tissue. Where tested, a corresponding increase in CDP/Cux stable DNA binding activity was observed. DNA sequencing analysis of CUTL1 cDNAs from 6 leiomyomas, including 4 with LOH of CUTL1, did not reveal any gross rearrangement or point mutations. Altogether these findings suggest that CUTL1 is probably not the tumor suppressor on 7q22. Moreover, the frequent increase in smaller CDP/Cux isoforms indicates that molecular events associated with the truncation of CDP/Cux proteins may be selected in uterine leiomyomas.


Subject(s)
Gene Deletion , Leiomyoma/genetics , Nuclear Proteins/genetics , Repressor Proteins/genetics , Uterine Neoplasms/genetics , Female , Homeodomain Proteins , Humans , Loss of Heterozygosity , Nuclear Proteins/biosynthesis , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Repressor Proteins/biosynthesis , Transcription Factors
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