EP2445500A1 - Polythérapie à base d'un inhibiteur du point de contrôle g2 (sb-218078) et d'un agent chimiothérapeutique pour des cellules oncogènes dans des tumeurs solides - Google Patents

Polythérapie à base d'un inhibiteur du point de contrôle g2 (sb-218078) et d'un agent chimiothérapeutique pour des cellules oncogènes dans des tumeurs solides

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Publication number
EP2445500A1
EP2445500A1 EP10737760A EP10737760A EP2445500A1 EP 2445500 A1 EP2445500 A1 EP 2445500A1 EP 10737760 A EP10737760 A EP 10737760A EP 10737760 A EP10737760 A EP 10737760A EP 2445500 A1 EP2445500 A1 EP 2445500A1
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Prior art keywords
cells
chemotherapeutic agent
checkpoint inhibitor
cisplatin
treatment
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EP10737760A
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German (de)
English (en)
Inventor
De Maria Ruggero
Monica Bartucci
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Istituto Superiore di Sanita ISS
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Istituto Superiore di Sanita ISS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4535—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom, e.g. pizotifen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention is concerned with the use of a G2 checkpoint inhibitor and a chemotherapeutic agent in the manufacture of a medicament for the treatment of tumorigenic cells in solid tumours. Also provided is a pharmaceutical pack comprising same, the same for the treatment of tumorigenic cells in solid tumours, and methods of treatment of tumorigenic cells in solid tumours using same.
  • tumours arise, spread, acquire resistance to anti-neoplastic drugs and ultimately relapse because they are sustained by a population of undifferentiated cells (1-15; references are detailed at the end of the description, immediately prior to the claims). These cells are endowed with stem cell-like properties of self-renewal and multipotency since they are able to give rise to the differentiated non-tumorigenic bulk tumour population.
  • stem cells known as cancer stem cells (CSCs) or tumour initiating cells
  • CSCs cancer stem cells
  • tumour initiating cells represent only a small population in the tumour mass, they are necessary and sufficient for tumour development and maintenance (4).
  • the different sensitivity to anticancer agents of stem and differentiated cancer cell populations might account for the inability of current therapies to eradicate solid tumours (16).
  • Cancer stem cells are slow-dividing cells that have an unlimited proliferative potential. They have a high expression of anti-apoptotic proteins that renders them resistant to conventional therapies (16), thus resulting in the disease relapse that follows the treatment of many common cancers. It is also likely that conventional chemotherapy actually enriches CSCs allowing repopulation of the tumour mass. Bao et a/. (17) have demonstrated that glioma stem cells identified in CD133 positive cells possess a more functional DNA damage pathway than CD133-negative tumour cells, as a result they are more resistant to radiation therapy. It is therefore desirable to provide improved treatments for tumorigenic cells, particularly in solid tumours.
  • the present inventors have now found that the combination of a G2 checkpoint inhibitor, particularly a G2 checkpoint abrogator, together with a chemotherapeutic agent is surprisingly effective in the treatment of tumorigenic cells (also referred to as cancer stem cells; CSCs), and particularly so in solid tumours.
  • a G2 checkpoint inhibitor particularly a G2 checkpoint abrogator
  • chemotherapeutic agent is surprisingly effective in the treatment of tumorigenic cells (also referred to as cancer stem cells; CSCs), and particularly so in solid tumours.
  • CSCs cancer stem cells
  • a G2 checkpoint inhibitor and a chemotherapeutic agent in the manufacture of a medicament for the treatment of tumorigenic cells in solid tumours.
  • Also provided according to the present invention is a G2 checkpoint inhibitor and a chemotherapeutic agent for the treatment of tumorigenic cells in solid tumours.
  • a pharmaceutical pack for the treatment of tumorigenic cells in solid tumours comprising a G2 checkpoint inhibitor and a chemotherapeutic agent.
  • the G2 checkpoint inhibitor and the chemotherapeutic agent may be provided in a single combined formulation, or may be provided separately for discrete administration either separately or sequentially.
  • further agents for the maintenance and/or restoration of health such as an at least one further chemotherapeutic agent may be provided in any formulation or pharmaceutical pack.
  • Such further agents may include agents designed to cure, alleviate, remove or lessen the symptoms of, or prevent or reduce the possibility of contracting any disorder or malfunction of the human or animal body.
  • the use of a G2 checkpoint inhibitor together with a chemotherapeutic agent can result in a substantially enhanced efficacy in treating tumorigenic stem cells.
  • the G2 checkpoint inhibitor is a G2 checkpoint abrogator.
  • the G2 checkpoint inhibitor is a Chk1 inhibitor.
  • the G2 checkpoint inhibitor is a Chk1 protein inhibitor.
  • the G2 checkpoint inhibitor is a WEE1 inhibitor, more preferably PD0166285 (Pfizer (Ann Arbor, Ml, USA)).
  • the G2 checkpoint inhibitor is:
  • G2 checkpoint inhibitor is:
  • salts are pharmaceutically acceptable salts.
  • chemotherapeutic agents are suitable for use in the present invention.
  • the chemotherapeutic agent is an antineoplastic agent.
  • chemotherapeutic agents include Cisplatin, Gemcitabine and Paclitaxel.
  • Other suitable chemotherapeutic agents will be readily apparent to a person of ordinary skill in the art.
  • the tumorigenic cells are selected from the group consisting of: colon cancer stem cells, and lung cancer stem cells.
  • Other suitable tumorigenic cells will be readily apparent to one of ordinary skill in the art.
  • Also provided according to the present invention is method of treatment of tumorigenic cells in solid tumours, comprising the step of administering a G2 checkpoint inhibitor and a chemotherapeutic agent to a patient in need of same.
  • the present inventors have isolated cancer stem cells from lung cancer specimens of patients who underwent surgical resection of the tumour.
  • In vitro exposure of LCSCs to conventional chemotherapy resulted in a selective G2 phase arrest providing this population with the opportunity to repair the DNA after the drug- induced damage and cell survival.
  • the G2 checkpoint effectors are ATM and ATR kinases and the downstream Chk1 kinase (18).
  • Chk1 maintains the phosphorylation of Cdc2 protein kinase, whose activity, together with Cyclin B1 , is required for entry into mitosis (19, 20).
  • Cdc2 is inactivated and cells are arrested at checkpoints until damaged DNA is repaired, avoiding propagation of genetic defects to the daughter cells.
  • Chk1 inhibitors (SB-218078 and AZD7762) were used either alone or in combination with antineoplastic drugs currently in use in clinical practice.
  • the experimental results show that in lung cancer stem cells, G2 abrogators increased cytotoxicity of DNA damaging agents.
  • Combination therapy studies have also been successfully carried out in vivo.
  • Chk1 inhibitors significantly enhance the anti-tumour efficacy of Gemcitabine in mice xenograft tumour model at well tolerated doses.
  • an at least one further therapeutic agent may be provided or administered as appropriate.
  • an at least one further therapeutic agent is a chemotherapeutic agent.
  • cancer stem cells are slow-dividing cells that have an unlimited proliferative potential and are resistant to chemotherapy, this understanding being in line with the idea that this population might indeed account for treatment failure.
  • G2 checkpoint inhibitors are best administered after treatment with chemotherapeutic drugs (e.g. anti- neoplastic drugs).
  • chemotherapeutic drugs e.g. anti- neoplastic drugs.
  • the further experiments show that a concomitant (i.e. simultaneous or concurrent) administration of G2 checkpoint inhibitors, particularly Chk1 inhibitors, and chemotherapeutic agents is less effective (Figure 9).
  • Optimal administration regimes can be readily determined by undertaking further experiments in which administration times are varied, Further optimization of administration regimes (for example according to gender, age, or ethnic origin) can be achieved with appropriate in vivo and in vitro assays.
  • the administration pattern of the medicament is that the G2 checkpoint inhibitor is administered at least 30 minutes after the chemotherapeutic agent is administered. Preferably, it is administered at least 1 hour after the chemotherapeutic agent. Preferably, it is administered at least 2 hours after the chemotherapeutic agent. Preferably, it is administered at least 3 hours after the chemotherapeutic agent. Preferably, it is administered at least 4 hours after the chemotherapeutic agent. Preferably, it is administered at least 5 hours after the chemotherapeutic agent. Preferably, it is administered at least 6 hours after the chemotherapeutic agent.
  • chemotherapeutic agent is administered at least 7 hours after the chemotherapeutic agent.
  • it is administered at least 8 hours after the chemotherapeutic agent.
  • it is administered between about one half and 8, 1 and 8, 2 and 8, 3 and 8, 4 and 8, 5 and 8, 6 and 8, or 7 and 8 hours after the chemotherapeutic agent.
  • administration times can be considered to be times for administration or times at which administration is to take place.
  • co-administration of AZD7762 and Cisplatin yield the same outcome as co-administration of AZD7762 and Gemcitabine, namely significantly affected growth of human lung carcinoma generated by subcutaneous transplantation of LCSCs into NOD-SCID mice ( Figure 10).
  • Chk1 is indeed a successful target in the treatment of cancer, its chemical inhibition leading to tumorigenic cell population exhaustion in solid tumours.
  • G2 checkpoint inhibitors particularly Chk1 inhibitors, should be administered shortly after chemotherapy, preferably in a range of 7-8 hours.
  • Figure 1 A shows cell count after treatment.
  • Y-axis shows count (ranges: top left 0 -
  • X-axis shows Pl-A (top and centre) and Pi PE-A (bottom), scale identical on all graphs.
  • X-axis markings are (left to right) 10 ⁇ 2, 10 ⁇ 3, 1OM and 10 ⁇ 5;
  • Figure 1 B shows Western blot results of treatment of LCSC18, LCSC34 and
  • Figure 1C shows cell count after treatment.
  • Y-axis shows count (ranges: top left 0 - 8200; bottom left 0 - 680; top right 0 - 3000; bottom right 0 - 350).
  • X-axis shows Pl-A (ranges: top 0 - 260; bottom 0 - 150);
  • Figure 1 D shows viability of lung cancer stem cell cloned cells.
  • Y-axis shows cell viability (%) (range: 0 - 100).
  • X-axis columns are (left to right): NT, SB-218078,
  • LCSC34 top right
  • LCSC36 bottom left
  • LCSC136 bottom right
  • Figure 2C shows gels of (left) LCSC18, (centre) LCSC34 and (right) differentiated
  • LCSC18 for Survivin top
  • beta actin bottom
  • Cells were treated with (left to right): control, SB-218078, Cisplatin, and Cisplatin + SB-218078.
  • Figure 3A shows Caspase-3/7 activation of (left to right) LCSC18, LCSC34, and
  • LCSC136 Y-axes are Caspase-3/7 activation (fold increase vs. control), range
  • X-axes are (left to right): control, SB-218078, Cisplatin, Gemcitabine, Paclitaxel, Cisplatin + SB-218078, Gemcitabine + SB-218078, and Paclitaxel +
  • Figure 3B shows confocal microscopy of LCSC18 cells. Columns are (left to right) staining with TUNEL, staining with DPI, merged staining, and phase contrast microscopy. Intensity of staining with TUNEL and DAPI is tabulated beneath;
  • Figure 4A shows confocal microscopy of lung cancer stem cells. Left columns are staining with DAPI, centre columns are staining for Phalloidin, right columns are merge. Rows are (top to bottom): control, SB-218078, Paclitaxel, and Paclitaxel + SB-218-78;
  • Figure 4B shows bar charts of number of multinucleated cells for (left to right)
  • LCSC18, LCSC34 and LCSC136 clones Y-axis range is 0 - 100.
  • X-axis columns for each bar chart are (left to right): control, SB-218078, Cisplatin,
  • Figure 5A shows Western blot results of treatment of LCSC18 and LCSC34 with (left) LCSC18 and (right) LCSC34 treated with (left to right): control, SB- 218078, Cisplatin, Cisplatin + SB-218-78, Gemcitabine, and Gemcitabine + Sb-
  • Figure 5B shows confocal microscopy of LCSC34 cells. Columns are (left to right) staining for cyclin B1 , staining with TO-PRO-3, merged staining, and phase contrast microscopy. Results show that the extent of staining is: Cisplatin > Cisplatin + SB-218078 > SB-218078 > control.
  • Figure 6A shows cells analysed to determine the fractions of nectoric vc. apoptotic cells.
  • Y-axis on all plots is Pl A.
  • X-axes are (top row and bottom left) Annexin V Alexa Fluor 647 and (other plots) APC-A.
  • Caspase-2 (left four columns) Caspase-2, and (right four columns) Caspase-3. Each set of columns is (left to right) NT, SB-218078, Cisplatin, and Cisplatin + SB-218078.
  • X-axis range is 0 - 50;
  • Figure 6C shows average numbers of colonies per well for each indicated condition. Mean ⁇ SD of 3 independent experiments performed on 3 different
  • LCSC lines is shown.
  • Y-axis is number of colonies / well, range is (left and centre) 0 - 100, (right) 0 - 175.
  • Columns on left and centre bar charts are (left to right): Control, SB-218078, Cisplatin, Cisplatin + SB-218078.
  • Columns on right bar chart are (left to right): Control, SB-218078, AZD7762, Cisplatin, Cisplatin + SB-218078, Cisplatin + AZD7762;
  • Figure 7A shows tumour size (volume cm 3 ) in mouse xenograft models.
  • Y-axis is tumour size.
  • X-axis is number of days. Lines are (top to bottom): control, Gemcitabine, AZD7762, and AZD7762 + Gemcitabine;
  • Figure 7B shows a bar chart of tumour masses.
  • Y-axis is tumour mass (g), range 0 - 1.8g.
  • Columns are (left to right): control, Gemcitabine, AZD7762, and AZD7762 + Gemcitabine;
  • Figure 7C shows FACS analysis of human origin of tumours.
  • Y-axis is murine CD45 (moCD45 PE:Cy5-A).
  • X-axis is HLA (HLA I PE-A);
  • Figure 8 shows Western blots for p-Chk1 in LCSC#1 (LCSC34) and LCSC#2
  • Figure 9 shows viability of LCSCs after (i) concomitant (simultaneous) administration of Chk1 inhibitor and chemotherapeutic agents, and (ii) administration of Chk1 inhibitor 8 hours after chemotherapeutic agents.
  • X-axis indicates (left to right): NT - control (no treatment); SB (SB-218078 only); Cis (Cisplatin only); Cis+SB (Cisplatin + SB-218078); Paclitaxel (Paclitaxel only);
  • Pacli+SB (Paclitaxel + SB-218078). Y-axis shows cell viability (%);
  • Figure 10A shows effect of treatment of tumours in NOD-SCID mice with control (squares), AZD7762 (triangles), Cisplatin (circles) and cisplatin + AZD7762 (diamonds).
  • X-axis is number of days.
  • Y-axis is average tumour size (volume in cm 3 );
  • Figure 1OB shows average mass of tumours explanted from the mice used for Figure 1OB.
  • X-axis is (left to right): control, AZD7762, Cisplatin, and Cisplatin + AZD7762.
  • Y-axis is tumour mass (g);
  • FIG 11A shows carcinoembryonic antigen (CEA) levels in mice blood serum.
  • CEA carcinoembryonic antigen
  • FIG 11 B shows photographs of immunohistochemistry performed on formalin- fixed paraffin-embedded tissue for phosphorylated H2A.X (pH2A.X) from mice subjected to various treatments. Treatments are (left to right) Cisplatin; Cisplatin + AZD7762, Gemcitabine; and Gemcitabine + AZD7762. Second row of photographs are enlarged areas indicated in first row of photographs. The table indicates the number of positive p-H2A.X cells visible in the photographs; and Figure 11 C shows colony forming ability of recovered tumour cells. X-axis indicates (left to right): control; AZD7762; Cisplatin; Cisplatin + AZD7762; Gemcitabine; Gemcitabine + AZD7762.
  • LCSCs lung cancer stem cells
  • Recovered cells were cultured at clonal density in serum-free medium containing 50 mg/ml insulin, 100 mg/ml apo- transferrin, 10 mg/ml putrescine, 0.03 mM sodium selenite, 2 mM progesterone, 0.6% glucose, 5mM HEPES, 0.1% sodium bicarbonate, 0.4% BSA, glutamine and antibiotics, dissolved in DMEM-F12 medium (Gibco-lnvitrogen) and supplemented with 20 mg/ml EGF and 10 mg/ml bFGF. Flasks non-treated for tissue culture were used to reduce cell adherence and support growth as undifferentiated tumour spheres.
  • the medium was replaced or supplemented with fresh growth factors twice a week until cells started to grow forming floating aggregates.
  • Cultures were expanded by mechanical dissociation of spheres, followed by re-plating of both single cells and residual small aggregates in complete fresh medium. In these conditions, cancer stem cells are enriched and grow in suspension as spheres. This culture system has been shown to maintain both sternness and tumorigenic properties (14, 15). Cell cycle analysis. After treatment with different drug combinations, cells were collected, washed with PBS and dissociated with trypsin.
  • Cells were then incubated with a propidium iodide staining solution (trisodium citrate 0.1%, NaCI 9.65mM, NP40 0.3%, Pl 50 ⁇ g/ml, RNase A 200 ⁇ g/ml) for 30 minutes at room temperature in the dark. Cell cycle profile was analyzed by Fluorescence-activated cell sorting (FACS).
  • a propidium iodide staining solution trisodium citrate 0.1%, NaCI 9.65mM, NP40 0.3%, Pl 50 ⁇ g/ml, RNase A 200 ⁇ g/ml
  • Cell viability assay Cell viability was measured with colorimetric MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay or CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to standard protocols. Lung cancer stem cells were seeded at 5000 cells/well in triplicates in a 96-well plate and treated with Cisplatin 5ug/ml, Gemcitabine 25OuM, Paclitaxel 30ng/ml and SB-2181078 2OnM. Cell viability was analyzed by Victor 2 plate reader equipment (Wallac, Turku, Finland).
  • Pl/Annexin V staining was used to determine the fractions of apoptotic versus necrotic cells. A total of 50000 treated cells were washed with Annexin V-binding buffer (2.5mM CaCI 2 , 14OmM NaCI, 1OmM HEPES) and thereafter incubated with Annexin V Alexa Fluor 647 1.25 nM (Invitrogen), for 15 minutes at room temperature. Cells were again washed with Annexin V-binding buffer, re-suspended in buffer containing 5 ⁇ g/ml propidium iodide (Pl) and analysed by FACS.
  • Annexin V-binding buffer 2.5mM CaCI 2 , 14OmM NaCI, 1OmM HEPES
  • Annexin V Alexa Fluor 647 1.25 nM Invitrogen
  • Caspase Assay Lung cancer stem cells were seeded at 5000 cells/well in triplicates in a 96-well plate and treated with Cisplatin 5ug/ml (Teval), Gemcitabine 25OuM (Lilly), Paclitaxel 30ng/ml (Sigma) and SB-2181078 2OnM (Calbiochem). Caspase 3/7 activation was evaluated after 96h by Apo1 Caspase-3/7 Assay kit (Promega). Colorimetric or fluorimetric assays were analyzed by Victor 2 plate reader (Wallac, Turku, Finland).
  • Apoptosis Assay - TUNEL The cells were cyto-spun onto glass slides, fixed with 4% buffered paraformaldehyde and then permeabilized with 0.1% Triton X-100. Apoptosis was determined with the In Situ Cell Death Detection kit, Fluorescein (Roche), following the manufacturer's instructions. The cells containing DNA strand breaks were stained with fluorescein-dUTP, detected and counted by fluorescence microscopy.
  • Treated cells were cyto-spun onto glass slides, fixed with 4% buffered paraformaldehyde and then permeabilized with 0.1% Triton X-100. The slides were incubated over night with anti-phosphoH2A.X 2 ⁇ g/ml (Upstate), anti-cyclin B1
  • Soft agar assay Lung cancer stem cells were treated with Cisplatin (5 ⁇ g/ml), Paclitaxel (30ng/ml), SB-218078 (2OnM), AZD7762 (5nM) or in combinations of drug and inhibitor (SB-218078 and AZD7762 were added after 8 hours). Thereafter, 1000 cells were immediately plated in the top agar layer in each well of a 24-well culture plate with 0.27% top agar layer and 0.4% bottom agar layer (SeaPlaque Agarose, Cambrex). Both layers of the soft agar contained either Cisplatin (5 ⁇ g/ml), Paclitaxel (30ng/ml), SB-218078 (2OnM), AZD7762 (5nM) or combinations of treatment. Cultures were incubated at 37 C C for 20 days. Colonies from triplicate wells were stained with crystal violet (0.01% in 10% MetOH), visualized and counted under microscope and photographed with a Nikon D80 camera.
  • LCSCs Differentiation of LCSCs. Lung cancer stem cells were plated in DMEM (Gibco) supplemented with 10% FBS (fetal bovine serum) over night for attachment of cells. DMEM was replaced with BEGM growth medium (Lonza) to facilitate the progress of differentiation and incubated for 72h. Differentiated cells could thereafter be maintained in DMEM supplemented with 10% FBS.
  • DMEM Gibco
  • FBS fetal bovine serum
  • mice Female NOD-SCID mice were purchased from Charles River. All procedures were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and within the protocols approved by the lstituto Superiore di Sanita. Tumour stem cells were dissociated, counted and resuspended in a mix of sterile PBS and Matrigel (1 :1). 50000 lung cancer stem cells (LCSCs) were implanted s.c. (subcutaneously) into the right flank of each mouse in a volume of 0.1 to 0.2 mL using a 25-gauge needle. Tumours were allowed to grow to the designated size of 100 to 200 mm 3 before the administration of compound. Animals were dosed i.v.
  • LCSCs lung cancer stem cells
  • tumour growth was evaluated before every single administration. After 30 days, tumours were removed, weighted and dissociated to analyze the percentage of human cells vs. murine cells using a PE conjugated anti-human HLA1 antibody (eBioscience) and an a PE-Cy5 anti-mouse CD45 antibody (BD Pharmingen) by FACS analysis.
  • a PE conjugated anti-human HLA1 antibody eBioscience
  • BD Pharmingen BD Pharmingen
  • G2 abrogators increase the cytotoxicity of DNA damaging agents in cancer stem cells
  • SB-218078 increases the cytotoxicity of Cisplatin, Gemcitabine and Paclitaxel on lung cancer stem cells and chemotherapy induces activation of Chk1.
  • Figure 1A Lung cancer stem cells were seeded at 250000/ml and treated with Cisplatin (5 ⁇ g/ml), Gemcitabine (250 ⁇ M) and Paclitaxel (30ng/ml) for 96h. Control and chemotherapy-treated cells were incubated with 5 ⁇ g/ml propidium iodide (Pl) and analyzed by FACS to determine the cell viability. Results are shown in Figure 1A.
  • Figure 1 B Lung cancer stem cells were seeded at 250000/ml and treated with Cisplatin (5 ⁇ g/ml), Gemcitabine (250 ⁇ M) and Paclitaxel (30ng/ml) for 96h. Control and chemotherapy-treated cells were incubated with 5 ⁇ g/ml propidium iodide (Pl) and analyzed by FACS to determine the cell viability. Results are shown in Figure 1A.
  • LCSC18 LCSC34 and LCSC36 were treated with Cisplatin (5 ⁇ g/ml), Gemcitabine (250 ⁇ M) and Paclitaxel (30ng/ml) for 96h.
  • Western blot was performed with 20 ⁇ g of whole cell protein extracts. Membranes were incubated with the antibodies specific for phosphorylated chk1 (pchki). Anti- ⁇ -actin antibody was used to assess equal loading. Results are shown in Figure 1 B and the table is indicating the relative expression of proteins analyzed.
  • FIG. 1 C LCSC18 were seeded at 2500007ml and treated with Cisplatin and Paclitaxel as described. After 48 hours of treatment, cells were incubated with a propidium iodide staining solution (trisodium citrate 0.1%, NaCI 9.65mM, NP40 0.3%, Pl 50 ⁇ g/ml, RNase 200 ⁇ g/ml) for 30 min at room temperature. Cell cycle was analyzed by FACS. Results are shown in Figure 1C.
  • Figure 1 D Lung cancer stem cell clones 18, 34, 36 and 136 were seeded at 5000 cells/well in triplicates in a 96-well plate and treated with Cisplatin (5 ⁇ g/ml), Gemcitabine (250 ⁇ M) or Paclitaxel (30ng/ml) for 96h and treated with or without SB- 218078 (2OnM) after 8 hours of the total incubation time. Cell viability in control and chemotherapy-treated cells was evaluated after 4 days by MTT assay and cell count. Data are a mean of three independent experiments. Results are shown in Figure 1 D.
  • Lung cancer stem cells resistance to chemotherapy relies on over-expression of anti-apoptotic proteins and a functional DNA damage pathway.
  • the first step in the initiation of activity of DNA damage checkpoint is recognition of DNA damage.
  • One of the earliest modifications of the chromatin structure in the damage response is phosphorylation of H2A.X at Ser 139 (29).
  • investigations were undertaken to determine whether exposure to Cisplatin or Paclitaxel would induce DNA damage in lung cancer stem cells, thus indicating the presence of DNA double strand breaks (DSBs) and the activation of the DNA damage machinery.
  • Lung cancer stem cells and differentiated cancer cells posses different repair and survival systems.
  • FIG. 2A LCSC34 were treated with Cisplatin and Cisplatin + SB-218078 for 6h and 96h. Cells were cyto-spun on glass slides, fixed with 4% buffered paraformaldehyde and then permeabilized with 0.1% Triton X-100. The slides were incubated overnight with anti-phospho-H2A.X, and then with an Alexa Fluor 647 secondary antibody for 1h at room temperature. DAPI was used to visualize nuclei. The slides were analyzed by confocal microscopy using an inverted fluorescence microscope (Olympus). Original magnification 6Ox.
  • FIG. 2B Differentiated lung cancer stem cells were seeded at 5000 cells/well in triplicates in a 96-well plate and treated with Cisplatin (5 ⁇ g/ml), Gemcitabine (250 ⁇ M) or Paclitaxel (30ng/ml) for 96h and treated with or without SB-218078 (2OnM) after 8 hours of the total incubation time. Cell viability in control and chemotherapy-treated cells was evaluated after 4 days by CellTiter-Glo assay.
  • FIG. 2C LCSC18 and LCSC34 were treated with Cisplatin (5 ⁇ g/ml), SB-218078 (2OnM) and Cisplatin + SB-218078 for 96h.
  • Western blot was performed with 20 ⁇ g of whole cell protein extracts. Membranes were incubated with the antibody specific for Survivin. The anti- ⁇ -actin antibody was used to assess equal loading. Table is indicating the relative expression of proteins analyzed.
  • Survivin is a member of the anti-apoptotic proteins family and its function is to inhibit caspases activation leading therefore to negative regulation of apoptosis or programmed cell death.
  • the suppression of apoptosis is a major contributing factor to carcinogenesis. Allowing cells to bypass apoptosis and continue to live and proliferate allows the eventual accumulation of mutations that may render the cell unresponsive to mechanisms that normally regulate cell growth.
  • the anti-apoptotic function of Survivin has been shown to play a significant role in cancer progression. Survivin has been shown to be regulated during the cell cycle as its expression is found to be dominant only in the G2/M phase.
  • G2 abrogators induce aberrant mitosis in cancer stem cells.
  • Efficiency of action of DNA damaging agents used in anticancer therapy depends on their ability to induce growth arrest and to activate the cell death machinery.
  • Cell death can be classified according to its morphological appearance (which may be apoptotic, necrotic, autophagic or associated with mitosis), enzymological criteria (with and without the involvement of nucleases or proteases) and functional aspects (programmed or accidental, physiological or pathological) (30).
  • An analysis of the biochemical and morphological features of the cell model in the presence and in the absence of treatments was therefore undertaken.
  • Combination with SB-218078 does not induce apoptosis in lung cancer stem cells after 96h treatment.
  • FIG. 3A Lung cancer stem cells were seeded at 5000 cells/well in triplicates in a 96-well plate and treated with Cisplatin (5 ⁇ g/ml), Gemcitabine (250 ⁇ M) or Paclitaxel (30ng/ml) for 96h and treated with or without SB-218078 (2OnM) after 8 hours of the total incubation time.
  • Caspase-3/7 activation was evaluated after 4 days by Apo1 Caspase-3/7 Assay kit (Promega). Colorimetric or fluorimetric assays were analyzed by Victor 2 plate reader (Wallac, Turku, Finland). Data are a mean of three independent experiments.
  • FIG. 3B LCSC18 were treated as described for 96h. Cells were cyto-spun on glass slides, fixed with 4% buffered paraformaldehyde and then permeabilized with 0.1% Triton X-100. Occurrence of apoptosis was determined with the In Situ Cell Death Detection kit, Fluorescein (Roche), following the manufacturer's instructions. The cells containing DNA strand breaks were stained with fluorescein-dUTP and detected by fluorescence microscopy. The table is showing the number of apoptotic cells vs non apoptotic cells.
  • Cisplatin in combination with SB-218078 causes mitotic catastrophe in lung cancer stem cells.
  • Figure 4A Lung cancer stem cells were treated as described previously. After 96h, cells were washed, cyto-spun on glass slides and stained with Phalloidin and DAPI to visualize cell membranes and nuclei. The slides were analyzed by confocal microscopy using an inverted fluorescence microscope (Olympus).
  • Figure 4B Percentage of multinucleated LCSC18, LCSC34 and LCSC136 was estimated by counting the nuclei in 100 cells on each slide. Data are presented as a mean of three independent experiments.
  • Apoptosis is recognized as a major barrier that must be circumvented by tumour cells to allow them to survive and proliferate in such stressful conditions (31). Its induction by DNA damage is typically associated with activation of the caspase family of proteases, which eventually leads to biochemical and morphological apoptosis-specific changes such as cellular shrinkage, membrane blebbing and DNA fragmentation (32).
  • tumours can be effectively eliminated following DNA damage by necrosis, mitotic catastrophe and autophagy.
  • Death by autophagy is characterized by the double-membrane vesicles containing cytosolic organelles (33).
  • Cells dying from mitotic catastrophe are usually large and contain uncondensed chromosomes.
  • the main characteristic of mitotic catastrophe is the formation of multiple micronuclei, and also aberrant mitotic spindle formation can be involved (34).
  • mitotic catastrophe is the formation of multiple micronuclei, and also aberrant mitotic spindle formation can be involved (34).
  • necrosis cells swell and loose their membrane integrity (35).
  • Investigation of the changes in cell morphology after drug treatment showed the presence of a large population of cells with two or more nuclei in the combination Cisplatin and SB-218078, which was indicative of cell death through mitotic catastrophe ( Figure 4A and B).
  • G2 abrogators induce mitotic catastrophe through premature activation of cdc2/cyclin B1 complex.
  • the expression of cell-cycle regulatory proteins, namely cdc2 and cyclin B1 was analyzed.
  • FIG. 5A LCSC18 and LCSC34 were treated with Cisplatin (5 ⁇ g/ml), Gemcitabine (250 ⁇ M) for 96h and treated with or without SB-218078 (2OnM) after 8 hours of the total incubation time.
  • Western blot was performed with 20 ⁇ g of whole cell protein extracts. Membranes were incubated with the antibodies specific for phosphorylated cdc2 (pcdc2) and cyclin B1. The anti- ⁇ -actin antibody was used to assess equal loading. Table is indicating the relative expression of proteins analyzed.
  • FIG. 5B LCSC34 were treated with Cisplatin (5 ⁇ g/ml), SB-218078 (2OnM) and Cisplatin + SB-218078 for 96h.
  • Cells were cyto-spun on glass slides, fixed with 4% buffered paraformaldehyde and then permeabilized with 0.1% Triton X-100. The slides were incubated over night with anti-cyclin B1 , and then with an Alexa Fluor 488 secondary antibody for 1 h at room temperature.
  • TO-PRO-3 was used to visualize nuclei.
  • the slides were analyzed by confocal microscopy using an inverted fluorescence microscope (Olympus). Original magnification 4Ox.
  • Chk1 phosphorylates the family of Cdc25 phosphatases, which in turn inhibit Cdc2 activity and prevent its premature activation, thus maintaining the highly regulated temporal order of cell-cycle progression.
  • Chk1 is activated to phosphorylate Cdc25 leading to its degradation via the proteasomal pathway or sequestration to cytoplasm by binding to 14-3-3 protein (36, 37).
  • Cdc2 is inactivated and cells are arrested at checkpoints until damaged DNA is repaired. It is reasonable to think that under this circumstance, inhibition of Chk1 leads to the improper activation of Cdc2 resulting in checkpoint abrogation and mitotic catastrophe.
  • Cdc2 is therefore the major target of Chk1 and is essential for G1/S and G2/M phase transitions, while cyclin B1 is a regulatory protein involved in mitosis and it translocates from the cytoplasm to the nucleus during M phase.
  • cyclin B1 is a regulatory protein involved in mitosis and it translocates from the cytoplasm to the nucleus during M phase.
  • cdc2 forms a complex with cyclin B1
  • a dividing cell is allowed to enter mitosis from G2 phase.
  • Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. Results show that in the cells treated with Cisplatin alone, cyclin B1 is clearly located in the cytoplasm, as a sign of cell cycle arrest. In cells treated with Cisplatin and SB-218078, cyclin B1 translocates from the cytoplasm to the nucleus, demonstrating that SB-218078 abrogates the G2 arrest induced by Cisplatin and forces the cells back into the cell cycle (Figure 5B).
  • mitotic catastrophe is thought to directly cause cell death that is distinct from apoptosis (39) or, alternatively, mitotic catastrophe is inherently associated with activating apoptosis from the M phase (39).
  • mitotic catastrophe is not a distinct form of cell death but rather an event that leads to cell death through apoptosis and/or necrosis (38, 39). To distinguish between them and to investigate how severe the damage induced by the combination of anti-neoplastic drugs and SB-218078 was, rescue experiments were performed.
  • FIG. 6A The combination of Cisplatin and SB-218078 induces irreparable damage in lung cancer stem cells.
  • Lung cancer stem cells were seeded at 250000/ml and treated with Cisplatin (5 ⁇ g/ml), SB218078 (20ng/ml) and the combination of both for 96h. At the end of the incubation period, cells were washed and plated in serum free medium for a total period of 72h. A total of 50000 cells/condition were double stained for propidium iodide (Pl) and Annexin V Alexa Fluor 647 and analysed by FACS to determine the fractions of necrotic vs. apoptotic cells.
  • Pl propidium iodide
  • Annexin V Alexa Fluor 647 Annexin V Alexa Fluor 647
  • FIG. 6B Lung cancer stem cells LCSC36 were treated with Cisplatin (5 ⁇ g/ml), SB-218078 (2OnM) and the combination of both for 96h. At the end of the incubation period, cells were washed and plated in fresh serum free medium for a total period of 72h. Western blot was performed with 20 ⁇ g of whole cell protein extracts. Membranes were incubated with antibodies specific for the pro-forms of caspase-2 and caspase-3, described in the general methods. The anti- ⁇ -actin antibody was used to assess equal loading. The table is indicating the relative expression of inactive pro-forms of caspases 2 and 3.
  • FIG. 6C Cells were treated with Cisplatin (5 ⁇ g/ml) and SB-218078 (2OnM, added after 8 hours) and then immediately plated in the soft agar top layer. 1000 cells were plated in each well of a 24-well culture plate with 0.4% bottom agar layer and 0.27 % top agar layer.
  • the soft agar contained either SB-218078 (2OnM), Cisplatin (5 ⁇ g/ml) and AZD7762 (5nM), or their combinations. Cultures were incubated at 37°C for 20 days. Colonies were stained with crystal violet 0.01 %, visualized and counted under microscope.
  • FITC Annexin V Staining with FITC Annexin V is typically used in conjunction with a vital dye such as propidium iodide (Pl) to allow the investigators to identify early apoptotic cells (Pl negative, FITC Annexin V positive) from necrotic cells (both FITC Annexin V and Pl positive).
  • Pl propidium iodide
  • Caspase-3 is the common downstream effector caspase for both the extrinsic/death receptor- induced apoptosis pathway involving caspase-8, and the intrinsic/mitochondria- triggered apoptotic pathway involving caspase-9 (40, 41) and it resulted in strong activation in the combination treatment ( Figure 6B).
  • cell death occurring during the metaphase/anaphase transition is characterized by the activation of caspase-2 which can also be activated in response to DNA damage.
  • Caspase-2 appears to be necessary for the onset of apoptosis triggered by several insults, including DNA damage (42, 43). Although the morphological aspect of apoptosis may be incomplete, these alterations constitute the biochemical hallmarks of apoptosis.
  • the combination Cisplatin + SB-218078 is also characterized by the activation of caspase-2, confirming even more that final cell death occurs through apoptosis ( Figure 6B). These results indicated that SB-218078 potentiates the cytotoxicity of chemotherapeutic drugs in short term viability assays. To investigate the long term impact of the combination with SB-218078 in lung cancer stem cells, soft agar assays were performed to evaluate differences in colony forming abilities.
  • Combination of chemotherapy and chk1 inhibitors has strong affects on tumour growth in vivo.
  • Xenotransplantation of LCSCs provides a solid base for the clinic use of therapies which aim to neutralize the pathways promoting tumour survival and growth.
  • therapies which aim to neutralize the pathways promoting tumour survival and growth.
  • chk1 inhibitors to enhance cytotoxicity of chemotherapeutic agents in lung cancer in vivo, the effect of AZD7762 on human lung carcinoma generated by subcutaneous transplantation of lung cancer stem cells into NOD-SCID mice was assessed.
  • AZD7762 potentiates Gemcitabine in mouse xenograft efficacy models.
  • Figure 7A NOD-SCID mice bearing tumours obtained by injection of LCSC136 were treated with multiple cycles of therapy every 3d. Each cycle consisted of Gemcitabine alone (60 mg/kg) or Gemcitabine (60 mg/kg) followed by one dose of 10 mg/kg AZD7762 (8 h after Gemcitabine dose). Tumour volume was measured using a callipers on the indicated days. Each Group consisted of 5 animals.
  • FIG. 7B Tumour masses were evaluated by weighting the tumours right after explantation.
  • the graph shows the median weight value of tumours for each group of mice.
  • DNA damage checkpoints are mechanisms that coordinate the progression of cell cycle with intracellular and extracellular events.
  • DNA damage checkpoint an elaborate network of signalling pathways, collectively called DNA damage checkpoint, is activated to prevent the damaged DNA from being replicated or transmitted to the next generation.
  • DNA damage checkpoint responses therefore play essential roles in cellular radio and chemosensitivity.
  • Cancer stem cells contribute to tumour repopulation through preferential checkpoint response and DNA repair, and targeting of checkpoint response in cancer stem cells can overcome chemo-resistance and may provide a therapeutic advantage to reduce tumour recurrence.
  • chemotherapies and Chk1 inhibitors were used: cisplatin (5 ug/ml), gemcitabine (250 ⁇ M), paclitaxel (30 ng/ml), SB218078 (20 nM), AZD7762 (5 nM).
  • gemcitabine 60 mg/kg
  • cisplatin 3 mg/kg
  • AZD7762 10 mg/kg
  • Chk1 inhibitors for both, in vitro and in vivo studies was added 8 hours after chemotherapy.
  • LCSCs and differentiated counterparts were treated for 12 hours as previously described.
  • Whole cell lysates were prepared in lysis buffer (NP40 1%, 20 mM TRIS (pH 7.2), 200 mM NaCI, Phosphatase Inhibitor Cocktail 1 (used 1 :100, P2850, Sigma- Aldrich, St. Louis, MO, USA), Phosphatase Inhibitor Cocktail 2 (used 1:100, P5726, Sigma-Aldrich) and Protease Inhibitor Cocktail (used 1 :100, P8340, Sigma-Aldrich)).
  • lysis buffer NP40 1%, 20 mM TRIS (pH 7.2), 200 mM NaCI
  • Phosphatase Inhibitor Cocktail 1 used 1 :100, P2850, Sigma- Aldrich, St. Louis, MO, USA
  • Phosphatase Inhibitor Cocktail 2 used 1:100, P5726, Sigma-Aldrich
  • mice Female NOD-SCID mice were purchased from Charles River. All procedures were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and within the protocols approved by the lstituto Superiore di Sanita. LCSCs were dissociated, counted and resuspended in a mix of PBS and Matrigel (1 :1). 50,000 LCSCs were implanted subcutaneously into the right flank of each mouse in a volume of 0.1 to 0.2mL using a 25-gauge needle. Tumours were allowed to grow to the size of 100 to 200 mm 3 before the administration of compounds.
  • Tumour mass was analyzed by weighting tumours using a PL202-L Precision Balance (Mettler-Toledo, Novate Milanese Ml, Italy), lmmunohistochemistry was performed on formalin fixed paraffin-embedded tissue or frozen tissue. Paraffin sections (5 ⁇ m) were de-waxed in xylene and rehydrated with distilled water. The slides were subsequently incubated with phosphorylated H2A.X (Upstate-Millipore). The reaction was performed using Elite Vector Stain ABC systems (Vector Laboratories) and DAB substrate chromogen (DakoCytomation), followed by counterstaining with haematoxylin. The percentage of human cells vs. murine cells was evaluated by FACS analysis using a PE conjugated anti-human HLA1 antibody (eBioscience) and a PE-Cy5 anti-mouse CD45 antibody (BD Pharmingen).
  • Soft agar assay Soft agar colony forming assays were carried out for LCSCs treated with cisplatin, paclitaxel, SB218078, AZD7762 or in combinations of drug and inhibitor for 96 hours. Subsequently, cells were washed and 1 ,000 cells were plated in the top agar layer in each well of a 24-well culture plate with 0.3% top agar layer and 0.4% bottom agar layer (SeaPlaque Agarose, Cambrex, New Jersey, USA). Cultures were incubated at 37 C C for 20 days. Colonies from triplicate wells were stained with crystal violet (0.01% in 10% MetOH), visualized and counted under microscope and photographed with a Nikon D80 camera.
  • LCSCs are found to possess a much more efficient activation of
  • Chk1 (i.e. a more efficient DNA repair pathway) than differentiated counterparts.
  • LCSC#1 LCSC34
  • LCSC#2 LCSC36
  • cisplatin 5 ⁇ g/ml
  • paclitaxel 30ng/ml
  • SB-218078 SB-218078
  • Results show that Chk1 inhibitors work more effectively when administered after chemotherapeutic agents, i.e. that Chk1 inhibitors achieve enhanced efficacy when administered after chemotherapeutic agents. Further experiments were performed as above, with the G2 checkpoint inhibitor (i.e. the Chk1 inhibitor) administered 7 hours after the chemotherapeutic agent, and results (not shown) are generally the same as those shown for 8 hours.
  • the G2 checkpoint inhibitor i.e. the Chk1 inhibitor
  • Groups of NOD-SCID mice (five animals per group) were injected with LCSC136 to give tumours. They were then treated with multiple cycles of therapy every 3 days (first treatment is day 0). Each treatment cycle consisted of: Group (i) - control, no treatment; Group (ii) - AZD7762 alone (10 mg/kg); Group (iii) - Cisplatin alone (3mg/kg), or Group (iv) - Cisplatin (3mg/kg) followed by one dose of 10 mg/kg AZD7762 (8 hours after Cisplatin dose). Tumour volume was measured using an electronic caliper on the indicated days. Tumour mass was evaluated by weighing the tumour immediately after explantation at day 30.
  • Results show that co-administration of Cisplatin with AZD7762 strongly affects tumor growth in terms of growth rate and tumour mass.
  • FIG. 11 A Carcinoembryonic antigen (CEA) levels in mice blood serum were measured using an immunoluminometric technique. Mean value ⁇ 1 SD of three experiments is reported.
  • CEA Carcinoembryonic antigen
  • H2A.X phosphorylated H2A.X
  • Figure 11 B shows by immunohistochemical analysis of tumour xenograft tissues that combination treatment with Chk1 inhibitors is irreparably damaging tumour cells through an increased expression of pH2A.X.
  • Figure 11 C shows inhibition/reduction of clonogenic ability accounting for disposal of tumorigenic cells (CSCs) in the tumour xenograft.
  • Singh SK Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J et al. Identification of a cancer stem cell in human brain tumours. Cancer Res 2003; 63: 5821-5828. PMID: 14522905 8) Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T et al. Identification of human brain tumour initiating cells. Nature 2004; 432: 396-401. PMID: 15549107

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Abstract

L'invention concerne l'utilisation d'un inhibiteur de point de contrôle G2 tel que SB218078, PD0166285 ou AZD7762 et d'un agent chimiothérapeutique tel que cisplatine, paclitaxel et gemcitabine dans la fabrication d'un médicament destiné à traiter des cellules oncogènes dans des tumeurs solides. L'invention concerne aussi un ensemble pharmaceutique comprenant ce médicament et destiné à traiter des cellules oncogènes dans des tumeurs solides, et des méthodes de traitement de cellules oncogènes dans des tumeurs solides à l'aide dudit ensemble.
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