Publications by authors named "Clara Albanese"

16 Publications

  • Page 1 of 1

Cord blood-derived cytokine-induced killer cells combined with blinatumomab as a therapeutic strategy for CD19 tumors.

Cytotherapy 2018 08 7;20(8):1077-1088. Epub 2018 Aug 7.

Center of Cellular Therapy "G. Lanzani", Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy. Electronic address:

Background: Cytokine-induced killer cells (CIKs) are an advanced therapeutic medicinal product (ATMP) that has shown therapeutic activity in clinical trials but needs optimization. We developed a novel strategy using CIKs from banked cryopreserved cord blood units (CBUs) combined with bispecific antibody (BsAb) blinatumomab to treat CD19 malignancies.

Methods: CB-CIKs were expanded in vitro and fully characterized in comparison with peripheral blood (PB)-derived CIKs.

Results: CB-CIKs, like PB-CIKs, were mostly CD3 T cells with mean 45% CD3CD56 and expressing mostly TCR(T cell receptor)αβ with a TH1 phenotype. CB-CIK cultures had, however, a larger proportion of CD4 cells, mostly CD56, as well as a greater proportion of naïve CCR7CD45RA and a lower percentage of effector memory cells, compared with PB-CIKs. CB-CIKs were very similar to PB-CIKs in their expression of a large panel of co-stimulatory and inhibitory/exhaustion markers, except for higher CD28 expression among CD8 cells. Like PB-CIKs, CB-CIKs were highly cytotoxic in vitro against natural killer (NK) cell targets and efficiently lysed CD19 tumor cells in the presence of blinatumomab, with 30-60% lysis of target cells at very low effector:target ratios. Finally, both CB-CIKs and PB-CIKs, combined with blinatumomab, showed significant therapeutic activity in an aggressive PDX Ph CD19 acute lymphoblastic leukemia model in NOD-SCID mice, without sign of toxicity or graft-versus-host disease. The improved expansion protocol was finally validated in good manufacturing practice conditions, showing reproducible expansion of CIKs from cryopreserved cord blood units with a median of 28.8 × 10 CIK/kg.

Discussion: We conclude that CB-CIKs, combined with bispecific T-cell-engaging antibodies, offer a novel, effective treatment strategy for leukemia.
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http://dx.doi.org/10.1016/j.jcyt.2018.06.003DOI Listing
August 2018

Tumor Targeting with an isoDGR-Drug Conjugate.

Chemistry 2017 Jun 26;23(33):7910-7914. Epub 2017 May 26.

Dipartimento di Chimica, Università degli Studi di Milano, Via C. Golgi 19, 20133, Milano, Italy.

Herein we report the first example of an isoDGR-drug conjugate (2), designed to release paclitaxel selectively within cancer cells expressing integrin α β . Conjugate 2 was synthesized by connecting the isoDGR peptidomimetic 5 with paclitaxel via the lysosomally cleavable Val-Ala dipeptide linker. Conjugate 2 displayed a low nanomolar affinity for the purified integrin α β receptor (IC =11.0 nm). The tumor targeting ability of conjugate 2 was assessed in vitro in anti-proliferative assays on two isogenic cancer cell lines characterized by different integrin α β expression: human glioblastoma U87 (α β +) and U87 β -KO (α β -). The isoDGR-PTX conjugate 2 displayed a remarkable targeting index (TI=9.9), especially when compared to the strictly related RGD-PTX conjugate 4 (TI=2.4).
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http://dx.doi.org/10.1002/chem.201701844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488297PMC
June 2017

Synthesis and biological evaluation of RGD peptidomimetic-paclitaxel conjugates bearing lysosomally cleavable linkers.

Chemistry 2015 Apr 17;21(18):6921-9. Epub 2015 Mar 17.

Università degli Studi di Milano, Dipartimento di Chimica, Via C. Golgi, 19, 20133, Milan (Italy), Fax: (+39) 02-5031-4072.

Two small-molecule-drug conjugates (SMDCs, 6 and 7) featuring lysosomally cleavable linkers (namely the Val-Ala and Phe-Lys peptide sequences) were synthesized by conjugation of the αvβ3-integrin ligand cyclo[DKP-RGD]-CH2NH2 (2) to the anticancer drug paclitaxel (PTX). A third cyclo[DKP-RGD]-PTX conjugate with a nonpeptide "uncleavable" linker (8) was also synthesized to be tested as a negative control. These three SMDCs were able to inhibit biotinylated vitronectin binding to the purified αVβ3-integrin receptor at nanomolar concentrations and showed good stability at pH 7.4 and pH 5.5. Cleavage of the two peptide linkers was observed in the presence of lysosomal enzymes, whereas conjugate 8, which possesses a nonpeptide "uncleavable" linker, remained intact under these conditions. The antiproliferative activities of the conjugates were evaluated against two isogenic cell lines expressing the integrin receptor at different levels: the acute lymphoblastic leukemia cell line CCRF-CEM (αVβ3-) and its subclone CCRF-CEM αVβ3 (αVβ3+). Fairly effective integrin targeting was displayed by the cyclo[DKP-RGD]-Val-Ala-PTX conjugate (6), which was found to differentially inhibit proliferation in antigen-positive CCRF-CEM αVβ3 versus antigen-negative isogenic CCRF-CEM cells. The total lack of activity displayed by the "uncleavable" cyclo[DKP-RGD]-PTX conjugate (8) clearly demonstrates the importance of the peptide linker for achieving the selective release of the cytotoxic payload.
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http://dx.doi.org/10.1002/chem.201500158DOI Listing
April 2015

Mcl-1 antagonism is a potential therapeutic strategy in a subset of solid cancers.

Exp Cell Res 2015 Mar 5;332(2):267-77. Epub 2014 Dec 5.

Nerviano Medical Sciences S.r.l. - Oncology, Viale Pasteur 10, I-20014 Nerviano, Milan, Italy.

Cancer cell survival is frequently dependent on the elevated levels of members of the Bcl-2 family of prosurvival proteins that bind to and inactivate BH3-domain pro-apoptotic cellular proteins. Small molecules that inhibit the protein-protein interactions between prosurvival and proapoptotic Bcl-2 family members (so-called "BH3 mimetics") have a potential therapeutic value, as indicated by clinical findings obtained with ABT-263 (navitoclax), a Bcl-2/Bcl-xL antagonist, and more recently with GDC-0199/ABT-199, a more selective antagonist of Bcl-2. Here, we report study results of the functional role of the prosurvival protein Mcl-1 against a panel of solid cancer cell lines representative of different tumor types. We observed silencing of Mcl-1 expression by small interfering RNAs (siRNAs) significantly reduced viability and induced apoptosis in almost 30% of cell lines tested, including lung and breast adenocarcinoma, as well as glioblastoma derived lines. Most importantly, we provide a mechanistic basis for this sensitivity by showing antagonism of Mcl-1 function with specific BH3 peptides against isolated mitochondria induces Bak oligomerization and cytochrome c release, therefore demonstrating that mitochondria from Mcl-1-sensitive cells depend on Mcl-1 for their integrity and that antagonizing Mcl-1 function is sufficient to induce apoptosis. Thus, our results lend further support for considering Mcl-1 as a therapeutic target in a number of solid cancers and support the rationale for development of small molecule BH3-mimetics antagonists of this protein.
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http://dx.doi.org/10.1016/j.yexcr.2014.11.022DOI Listing
March 2015

A novel method using blinatumomab for efficient, clinical-grade expansion of polyclonal T cells for adoptive immunotherapy.

J Immunol 2014 Nov 29;193(9):4739-47. Epub 2014 Sep 29.

Centro Trapianto Midollo Osseo, USC Ematologia, Azienda Ospedaliera Papa Giovanni XXIII, 24127 Bergamo, Italy;

Current treatment of chronic lymphocytic leukemia (CLL) patients often results in life-threatening immunosuppression. Furthermore, CLL is still an incurable disease due to the persistence of residual leukemic cells. These patients may therefore benefit from immunotherapy approaches aimed at immunoreconstitution and/or the elimination of residual disease following chemotherapy. For these purposes, we designed a simple GMP-compliant protocol for ex vivo expansion of normal T cells from CLL patients' peripheral blood for adoptive therapy, using bispecific Ab blinatumomab (CD3 × CD19), acting both as T cell stimulator and CLL depletion agent, and human rIL-2. Starting from only 10 ml CLL peripheral blood, a mean 515 × 10(6) CD3(+) T cells were expanded in 3 wk. The resulting blinatumomab-expanded T cells (BET) were polyclonal CD4(+) and CD8(+) and mostly effector and central memory cells. The Th1 subset was slightly prevalent over Th2, whereas Th17 and T regulatory cells were <1%. CMV-specific clones were detected in equivalent proportion before and after expansion. Interestingly, BET cells had normalized expression of the synapse inhibitors CD272 and CD279 compared with starting T cells and were cytotoxic against CD19(+) targets in presence of blinatumomab in vitro. In support of their functional capacity, we observed that BET, in combination with blinatumomab, had significant therapeutic activity in a systemic human diffuse large B lymphoma model in NOD-SCID mice. We propose BET as a therapeutic tool for immunoreconstitution of heavily immunosuppressed CLL patients and, in combination with bispecific Ab, as antitumor immunotherapy.
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http://dx.doi.org/10.4049/jimmunol.1401550DOI Listing
November 2014

9-Fluorenone-2-Carboxylic Acid as a Scaffold for Tubulin Interacting Compounds.

Chempluschem 2013 Jul 13;78(7):663-669. Epub 2013 May 13.

Dipartimento di Chimica-INSTM-UdR, and Istituto di Scienze e tecnologie molecolari del CNR (CNR-ISTM), Università degli Studi di Milano, Via Golgi 19, 20133 Milano (Italy).

The introduction of a hydrophobic group at position 7 of 9-fluorenone-2-carboxylic acid generates new tubulin binders, the design of which is suggested by modeling studies. The synthesis is based on the use of 2,7-dibromo-fluorenone as starting material. The antiproliferative activity on two different cell lines, fluorescent microscopy, flow cytometry, and sedimentation assay tests confirmed the supposed mechanism.
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http://dx.doi.org/10.1002/cplu.201300036DOI Listing
July 2013

The Polo-Like Kinase 1 (PLK1) inhibitor NMS-P937 is effective in a new model of disseminated primary CD56+ acute monoblastic leukaemia.

PLoS One 2013 8;8(3):e58424. Epub 2013 Mar 8.

Oncology, Nerviano Medical Sciences, Nerviano, Milano, Italy.

CD56 is expressed in 15-20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56(+) monoblastic AML (M5a). The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting (MST = 28, 36, 62 days for vehicle, cytarabine and NMS-P937, respectively). Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56(+) AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058424PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592825PMC
September 2013

Cdc7 kinase inhibitors: 5-heteroaryl-3-carboxamido-2-aryl pyrroles as potential antitumor agents. 1. Lead finding.

J Med Chem 2010 Oct;53(20):7296-315

Nerviano Medical Sciences Srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano, MI, Italy.

Cdc7 serine/threonine kinase is a key regulator of DNA synthesis in eukaryotic organisms. Cdc7 inhibition through siRNA or prototype small molecules causes p53 independent apoptosis in tumor cells while reversibly arresting cell cycle progression in primary fibroblasts. This implies that Cdc7 kinase could be considered a potential target for anticancer therapy. We previously reported that pyrrolopyridinones (e.g., 1) are potent and selective inhibitors of Cdc7 kinase, with good cellular potency and in vitro ADME properties but with suboptimal pharmacokinetic profiles. Here we report on a new chemical class of 5-heteroaryl-3-carboxamido-2-substituted pyrroles (1A) that offers advantages of chemistry diversification and synthetic simplification. This work led to the identification of compound 18, with biochemical data and ADME profile similar to those of compound 1 but characterized by superior efficacy in an in vivo model. Derivative 18 represents a new lead compound worthy of further investigation toward the ultimate goal of identifying a clinical candidate.
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http://dx.doi.org/10.1021/jm100504dDOI Listing
October 2010

Dual targeting of CDK and tropomyosin receptor kinase families by the oral inhibitor PHA-848125, an agent with broad-spectrum antitumor efficacy.

Mol Cancer Ther 2010 Aug 3;9(8):2243-54. Epub 2010 Aug 3.

Cell Biology Department, BU Oncology, Nerviano Medical Sciences, v.le Pasteur 10, Nerviano, Milan 20014, Italy.

Altered expression and activity of cyclin-dependent kinase (CDK) and tropomyosin receptor kinase (TRK) families are observed in a wide variety of tumors. In those malignancies with aberrant CDK activation, the retinoblastoma protein (pRb) pathway is deregulated, leading to uncontrolled cell proliferation. Constitutive activation of TRKs is instead linked to cancer cell survival and dissemination. Here, we show that the novel small-molecule PHA-848125, a potent dual inhibitor of CDKs and TRKs, possesses significant antitumor activity. The compound inhibits cell proliferation of a wide panel of tumoral cell lines with submicromolar IC(50). PHA-848125-treated cells show cell cycle arrest in G(1) and reduced DNA synthesis, accompanied by inhibition of pRb phosphorylation and modulation of other CDK-dependent markers. The compound additionally inhibits phosphorylation of TRKA and its substrates in cells, which functionally express this receptor. Following oral administration, PHA-848125 has significant antitumor activity in various human xenografts and carcinogen-induced tumors as well as in disseminated primary leukemia models, with plasma concentrations in rodents in the same range as those found active in inhibiting cancer cell proliferation. Mechanism of action was also confirmed in vivo as assessed in tumor biopsies from treated mice. These results show that the dual CDK-TRK inhibitor PHA-848125 has the potential for being a novel and efficacious targeted drug for cancer treatment.
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http://dx.doi.org/10.1158/1535-7163.MCT-10-0190DOI Listing
August 2010

Therapeutic efficacy of the pan-cdk inhibitor PHA-793887 in vitro and in vivo in engraftment and high-burden leukemia models.

Exp Hematol 2010 Apr 16;38(4):259-269.e2. Epub 2010 Feb 16.

Nerviano Medical Sciences Srl, Business Unit Oncology, Nerviano, Italy.

Objective: The aim of the work was to determine and characterize, in vitro and in vivo, the therapeutic activity of PHA-793887, a new potent pan-cdk inhibitor, in the context of hematopoietic neoplasms.

Materials And Methods: Thirteen leukemic cell lines bearing different cytogenetic abnormalities and normal hematopoietic cells were used in cytotoxicity and colony assays. The drug activity at the molecular level was analyzed by Western blotting. PHA-793887 was also tested in vivo in several leukemia xenograft models.

Results: PHA-793887 was cytotoxic for leukemic cell lines in vitro, with IC(50) ranging from 0.3 to 7 microM (mean: 2.9 microM), regardless of any specific chromosomal aberration. At these doses, the drug was not cytotoxic for normal unstimulated peripheral blood mononuclear cells or CD34(+) hematopoietic stem cells. Interestingly, in colony assays PHA-793887 showed very high activity against leukemia cell lines, with an IC(50) <0.1 microM (mean: 0.08 microM), indicating that it has efficient and prolonged antiproliferative activity. PHA-793887 induced cell-cycle arrest, inhibited Rb and nucleophosmin phosphorylation, and modulated cyclin E and cdc6 expression at low doses (0.2-1 microM) and induced apoptosis at the highest dose (5 microM). It was also effective in vivo in both subcutaneous xenograft and primary leukemic disseminated models that better mimic naturally occurring human disease. Interestingly, in one disseminated model derived from a relapsed Philadelphia-positive acute lymphoid leukemia patient, PHA-793887 showed strong therapeutic activity also when treatment was started after establishment of high disease burden.

Conclusions: We conclude that PHA-793887 has promising therapeutic activity against acute leukemias in vitro and in vivo.
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http://dx.doi.org/10.1016/j.exphem.2010.02.004DOI Listing
April 2010

Optimization of 6,6-dimethyl pyrrolo[3,4-c]pyrazoles: Identification of PHA-793887, a potent CDK inhibitor suitable for intravenous dosing.

Bioorg Med Chem 2010 Mar 25;18(5):1844-53. Epub 2010 Jan 25.

Nerviano Medical Sciences Srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano (MI), Italy.

We have recently reported CDK inhibitors based on the 6-substituted pyrrolo[3,4-c]pyrazole core structure. Improvement of inhibitory potency against multiple CDKs, antiproliferative activity against cancer cell lines and optimization of the physico-chemical properties led to the identification of highly potent compounds. Compound 31 (PHA-793887) showed good efficacy in the human ovarian A2780, colon HCT-116 and pancreatic BX-PC3 carcinoma xenograft models and was well tolerated upon daily treatments by iv administration. It was identified as a drug candidate for clinical evaluation in patients with solid tumors.
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http://dx.doi.org/10.1016/j.bmc.2010.01.042DOI Listing
March 2010

A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity.

Nat Chem Biol 2008 Jun 11;4(6):357-65. Epub 2008 May 11.

Nerviano Medical Sciences Oncology, Via Pasteur 10, 20014 Nerviano, Italy.

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.
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http://dx.doi.org/10.1038/nchembio.90DOI Listing
June 2008

6-Substituted pyrrolo[3,4-c]pyrazoles: an improved class of CDK2 inhibitors.

ChemMedChem 2007 Jun;2(6):841-52

Oncology Business Unit, Department of Chemistry, Nerviano Medical Sciences, Viale Pasteur 10, 20014 Nerviano MI, Italy.

We have recently reported a new class of CDK2/cyclin A inhibitors based on a bicyclic tetrahydropyrrolo[3,4-c]pyrazole scaffold. The introduction of small alkyl or cycloalkyl groups in position 6 of this scaffold allowed variation at the other two diversity points. Conventional and polymer-assisted solution phase chemistry provided a way of generating compounds with improved biochemical and cellular activity. Optimization of the physical properties and pharmacokinetic profile led to a compound which exhibited good efficacy in vivo on A2780 human ovarian carcinoma.
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http://dx.doi.org/10.1002/cmdc.200600302DOI Listing
June 2007

Cdc7 is an active kinase in human cancer cells undergoing replication stress.

J Biol Chem 2007 Jan 24;282(1):208-15. Epub 2006 Oct 24.

Department of Cell Biology, Nerviano Medical Sciences-Oncology, Via Pasteur 10, 20014 Nerviano, Italy.

Cdc7 kinase promotes and regulates DNA replication in eukaryotic organisms. Multiple mechanisms modulating kinase activity in response to DNA replication stress have been reported, supporting the opposing notions that Cdc7 either plays an active role under these conditions or, conversely, is a final target inactivated by a checkpoint response. We have developed new immnunological reagents to study the properties of human Cdc7 kinase in cells challenged with the ribonucleotide reductase inhibitor hydroxyurea or with the DNA topoisomerase II inhibitor etoposide. We show that Cdc7.Dbf4 and Cdc7.Drf1 complexes are stable and active in multiple cell lines upon drug treatment, with Cdc7.Dbf4 accumulating on chromatin-enriched fractions. Cdc7 depletion by small interfering RNA in hydroxyurea and etoposide impairs hyper-phosphorylation of Mcm2 at specific Cdc7-dependent phosphorylation sites and drug-induced hyper-phosphorylation of chromatin-bound Mcm4. Furthermore, sustained inhibition of Cdc7 in the presence of these drugs increases cell death supporting the notion that the Cdc7 kinase plays a role in maintaining cell viability during replication stress.
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http://dx.doi.org/10.1074/jbc.M604457200DOI Listing
January 2007

3-Aminopyrazole inhibitors of CDK2/cyclin A as antitumor agents. 2. Lead optimization.

J Med Chem 2005 Apr;48(8):2944-56

Department Chemistry, Nerviano Medical Sciences, BU-Oncology and BU-Preclinical Science, Viale Pasteur 10, 20014 Nerviano (MI), Italy.

Inhibitors of cyclin-dependent kinases (CDK) such as CDK2/cyclin A-E are currently undergoing clinical trials to verify their potential as new anticancer agents. In a previous article we described the lead discovery process of a 3-aminopyrazole class of CDK2/cyclin A-E inhibitors. The endpoint of this process was PNU-292137, a compound endowed with in vivo antitumor activity in a mouse tumor xenograft model. We optimized this lead compound to improve some physicochemical properties, notably solubility and plasma protein binding. This lead optimization process brought us to the discovery of (2S)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1-pyrrolidinyl)phenyl]propanamide (PHA-533533, 13), a compound with a balanced activity vs druglike profile. Compound 13 inhibited CDK2/cyclin A with a K(i) of 31 nM, counteracting tumor cell proliferation of different cell lines with an IC(50) in the submicromolar range. Solubility was improved more than 10 times over the starting lead, while plasma protein binding was decreased from 99% to 74%. With exploitation of this globally enhanced in vitro profile, 13 was more active than PNU-292137 in vivo in the A2780 xenograft model showing a tumor growth inhibition of 70%. Proof of mechanism of action was obtained in vivo by immunohistochemical analysis of tumor slices of 13-treated vs untreated animals.
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http://dx.doi.org/10.1021/jm0408870DOI Listing
April 2005

Cdc7 inhibition reveals a p53-dependent replication checkpoint that is defective in cancer cells.

Cancer Res 2004 Oct;64(19):7110-6

Department of Biology, Nerviano Medical Science, Nerviano, Italy.

Cdc7 is an evolutionarily conserved kinase that regulates S phase by promoting replication origin activation. Down-regulation of Cdc7 by small interfering RNA in a variety of tumor cell lines causes an abortive S phase, leading to cell death by either p53-independent apoptosis or aberrant mitosis. Unlike replication fork blockade, Cdc7-depleted tumor cells do not elicit a robust checkpoint response; thus, inhibitory signals preventing additional cell cycle progression are not generated. In normal fibroblasts, however, a p53-dependent pathway actively prevents progression through a lethal S phase in the absence of sufficient Cdc7 kinase. We show that in this experimental system, p53 is required for the lasting maintenance of this checkpoint and for cell viability. With this work we reveal and begin to characterize a novel mechanism that regulates DNA synthesis in human cells, and we suggest that inhibition of Cdc7 kinase represents a promising approach for the development of a new generation of anticancer agents.
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http://dx.doi.org/10.1158/0008-5472.CAN-04-1547DOI Listing
October 2004