WO2013071415A1 - Ciblage de la voie de rb destiné à la prévention du cancer - Google Patents

Ciblage de la voie de rb destiné à la prévention du cancer Download PDF

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WO2013071415A1
WO2013071415A1 PCT/CA2012/001058 CA2012001058W WO2013071415A1 WO 2013071415 A1 WO2013071415 A1 WO 2013071415A1 CA 2012001058 W CA2012001058 W CA 2012001058W WO 2013071415 A1 WO2013071415 A1 WO 2013071415A1
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inhibitor
cells
tumor
cancer
retinoblastoma
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Roderick Angus BREMNER
Sean R. McCURDY
Monika SANGWAN
Izhar LIVNE-BAR
Danian CHEN
Mohammad E. AHMAD
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University Health Network
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
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    • A61K31/425—Thiazoles
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    • 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
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • 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
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
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    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4736—Retinoblastoma protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to the prevention of cancer by targeting proteins in the RB pathway.
  • Cancer is a multi-step process that arises from continuous selection of more aggressive clones through sporadic mutagenesis.
  • Abnormal proliferation is a hallmark of cancer cells.
  • Most human cancers begin decades before they are detected, providing a long yet under-exploited latency period for prevention.
  • In the premalignant phase initiated cells can expand considerably, but remain contained either as benign tumors or as indistinct precancerous fields (Deng et al., 1996; Jonason et al., 1996; Moreno, 2008).
  • the number and type of oncogenic events required to reach this dangerous yet non-transformed "cancer-prone" state varies from tissue to tissue.
  • chemopreventative strategies are also critical for cancer survivors, who are at higher risk of secondary tumors due to pre-existing susceptibility and/or exposure to mutagenic treatments (Ng et al., 2010).
  • antiinflammatory drugs such as aspirin and non-steroidal anti-inflammatory drugs (NSAID) reduces cancer incidence (Kelloff et al., 2006).
  • NSAID non-steroidal anti-inflammatory drugs
  • E2f 1 , 2 and 3 Activating E2fs, (E2f 1 , 2 and 3) induce factors required for DNA replication, drive proliferation of quiescent cells (reviewed in (Cobrinik, 2005; Dimova and Dyson, 2005) and, although not required for normal progenitor division (Chen et al., 2009), are essential for abnormal division of differentiating Rb-nu ⁇ cells in multiple tissues (Chen et al., 2007; Chong et al., 2009; McClellan et al., 2007; Tsai et al., 1998; Ziebold et al., 2001).
  • the Rb-E2F pathway is a bistable switch that converts graded growth factor levels into all-or-none E2F responses that regulate progression into S-phase (Yao et al., 2008).
  • Cip/Kip inhibitors (p21 , p27 and p57) on the other hand bind and inhibit the kinase activity of Cyclin A/E-Cdk2/1 complexes.
  • Cdk2 inactivates the Rb family, and is essential to fire replication origins (Malumbres and Barbacid, 2009).
  • E2f and Cdk2 also work together to promote centrosome duplication (Meraldi et al., 1999). Inactivation of the Rb and CKI pathways and concomitant activation of E2f and Cdk occur together almost universally in human cancer (Malumbres and Barbacid, 2001).
  • the human retinoblastoma gene encodes a protein (RB) that is the pivotal component in a molecular pathway, which is defective in essentially all human cancers (Weinberg, 1995).
  • RB was the first recognized tumor suppressor gene, and was discovered through analysis of the childhood retinal cancer it is named after, retinoblastoma (DiCiommo et al. 2000).
  • the ocular cancer retinoblastoma generated fundamental discoveries with broad relevance to cell cycle regulation and cancer, including the classic two-hit hypothesis and RB, the first recognized tumor suppressor.
  • RB1 mutations are associated with >95% risk of retinal cancer (retinoblastoma) and 50% of RB *h survivors develop secondary tumors by the age of 50 (reviewed in Balmer et al., 2006).
  • retinoblastoma retinal cancer
  • 50% of RB *h survivors develop secondary tumors by the age of 50 (reviewed in Balmer et al., 2006).
  • one tumor arises from a developing retinal cell that spontaneously acquires defects in both copies of RB.
  • one gene copy is mutated in every cell (RB +/ ⁇ individuals), and the high probability of sustaining a defect in the second copy means that these children develop 5-12 tumors affecting both eyes.
  • the unique sensitivity of the human retina to RB loss implies that other human tissues and the retina in other species have extra protection.
  • p107 and p130 protect the mouse retina
  • current retinoblastoma models utilize the loss of Rb and one relative (Robanus-Maandag et al., 1998; Chen et al., 2004; MacPherson et al., 2004; Dannenberg et al., 2004; Zhang et al., 2004).
  • How p107lp130 protect the Rb '1' retina is unclear, but elucidating the mechanism could expose strategies to prevent tumors initiated by RB pathway defects in humans.
  • One explanation for the quantum difference between the tumor-resistant Rb '1' and tumor- prone Rb/p107-nu ⁇ retina is that the E2f targets become super-induced in the latter.
  • genetic screening follows presentation with retinoblastoma, usually in the first year of life. Positive identification of a germ line defect triggers intensive monitoring for new tumors. Effective strategies are required to prevent new tumors, either in the eye or in other organs later in life, or treat existing retinoblastoma tumors.
  • a method of preventing retinoblastoma in a subject comprising inhibiting the RB pathway.
  • an RB pathway inhibitor for the prevention of retinoblastoma in a subject.
  • an RB pathway inhibitor in the preparation of a medicament for the prevention of retinoblastoma in a subject.
  • an RB pathway inhibitor for use in the prevention of retinoblastoma in a subject is provided.
  • a method of determining the likelihood of a population of cells to be cancer-prone comprising: determining the level of E2f and Cdk activity in the population of cells, wherein a high level of both E2f and Cdk activity in the population compared to a control population is indicative of the population being cancer-prone.
  • a method of determining the likelihood of a population of cells to be cancer-prone comprising: determining the level of atypical protein kinase C activity in the population of cells, wherein a high or abnormal level of atypical protein kinase C in the population compared to a control population is indicative of the population being cancer-prone.
  • a method of determining the likelihood of a population of cells to be cancer-prone comprising: determining the level of Skp2 activity in the population of cells, wherein a high or abnormal level of both Skp2 activity in the population compared to a control population is indicative of the population being cancer-prone.
  • Figure 1 shows that both Rb-E2f and CKI-Cdk axes contribute to retinoblastoma initiation.
  • FIG. 2 shows that dividing Ap2a + amacrine like cells in multiple mouse models of retinoblastoma.
  • A Tumors of the indicated genotypes and ages were stained for ⁇ 2 ⁇ (green) and DAPI (blue). Scale bar 100 ⁇ .
  • B-D Tumors of the indicated genotypes and ages were stained for the cell cycle markers Ki67 (B), PH3 (C) and BrdU (D) (red), and the amacrine cell marker ⁇ 2 ⁇ (green). Scale bar 20 ⁇ .
  • Ki67 Ki67
  • C PH3
  • BrdU BrdU
  • amacrine cell marker ⁇ 2 ⁇ green
  • Scale bar 20 For simplicity "Rb'"' represents aCre;Rb m . See also Figure 8.
  • Figure 3 shows that Cdk activity correlates with tumor penetrance.
  • Figure 4 shows that p27, but not p21 , bound to Cdk2 correlates inversely with kinase activity and tumor penetrance.
  • A Anti-Cdk2 immunoprecipitates or straight lysates (input) from P8 retina of the indicated genotypes were immunoblotted for the proteins indicated on the left. Values below input blots represent protein expression as a percentage of Rb KO retina in a representative blot.
  • B-C Cdk2 kinase activity (from Figure 3) was plotted against the amount of bound p27 (B) or p21 (C) as determined in (A). "Rb ⁇ '" ⁇ s used to indicate aCre;Rb f/f . p values were calculated using a one-sample f-test for Pearson's product-moment correlational coefficient, r. All assays were run a minimum of 3 times.
  • FIG. 5 shows that heterozygosity for E2f1 is sufficient to block tumorigenesis in both Rb/p107 and Rb/p27 null retina.
  • P0 and adult (P30-P60) retinal sections of the indicated genotypes were stained for mitotic cells (PH3, red) and nuclei (DAPI, blue), or Hematoxylin and Eosin (H&E) (lower panels).
  • Apical mitoses (white arrows) represent normal progenitors whereas ectopic mitoses (red arrows) represent abnormally dividing differentiating neurons that are abundant in the Rblp107-nu ⁇ retina, and reduced or virtually absent when one or two E2f1 alleles are removed, respectively.
  • Scale bar 50 ⁇ .
  • FIG. 7 supplements Figure 1 and shows the expression of p21 and p27 at P8 in Wild Type (WT) and Rb null retina.
  • A Overlay of DAPI (blue) and anti-p21 (upper panel) or anti-p27 (lower panel) immunofluorescence (red) on horizontal sections from WT and Rb null retinas (O, ONL; I, INL; G, GCL).
  • Figure 8 supplements Figure 2 and shows that emerging Rb ⁇ ' ⁇ ;p2T' ⁇ tumors contain dividing cells positive for amacrine cell markers.
  • A Marker analysis of P30 WT and Rb '/' ;p2T /' retina. Low magnification views are shown on the left (scale bar 100 /m) and high magnification views of the boxed regions are shown on the right (scale bar 20 /m). Emerging tumors stained primarily for ⁇ 2 ⁇ (green), which specifically marks amacrine cells, Pax6 (red), which marks amacrine cells as well as other cell types, and i67 (green) which marks dividing cells.
  • Tumors had no or very few cells that stained for markers of cone, bipolar, ganglion or rod cells. Note also the absence of rods and cones in the thin untransformed KO retina (arrows), thus resembling the Rb/p107 null retina. All sections in A were also stained with DAPI to mark nuclei (blue). For simplicity "Rb ⁇ " represents oCre/R ⁇ .
  • Figure 9 supplements Table 1 and shows Loss of Heterozygosity (LOH) analysis in heterozygotes.
  • A Genotyping of tail (Tl) and tumor (Tm) DNA for p27° K ⁇ allele in aCre;Rb f/f ;p2T /CK ⁇ mice showing LOH as indicated by single 600bp band for p27° K' knock-in allele and loss of 520bp band for Wild Type (WT) allele in tumor DNA for all three tumor samples.
  • the p27 CK' diagnostic primers also often generate a background band between the 600 bp and 520 bp fragments.
  • Figure 10 supplements Figure 3 and shows that at P8 Cdk2 activity does not correlate with cell cycle index.
  • A P8 retinas of the indicated genotypes were stained for Ki67 (green) and DAPI (blue). Scale bar 50 //m.
  • B Quantification Ki67 positive cells in indicated genotypes. Data are mean ⁇ SD.
  • C Cdk2 kinase activity (percent of that in the Rb null retina) was plotted against percentage of ⁇ 67+ cells at P8. p value was determined using a one-sample t-test for Pearson's product-moment correlational coefficient, r. All assays were carried out at least 3 times. For simplicity "Rb-/-" represents aCre;Rbf/f.
  • Figure 11 supplements Figure 4 and shows that p21 levels bound to Cdk2 do not correlate with kinase activity.
  • Cdk2 kinase activity (from Figure 3) was plotted against the amount of p21 bound to Cdk2.”
  • Rb "/" is used to indicate oCre;Rb f f .
  • p values were calculated using a one-sample t-test for Pearson's product-moment correlational coefficient, r.
  • FIG. 12 shows Skp2 is essential for retinoblastoma initiation.
  • A Adult retinal sections stained with Hematoxylin and Eosin (H&E) at 10x and 4x of the indicated genotypes. Deletion of Skp2 completely blocked tumor initiation in the Rb/p107 DKO retina. Furthermore, Skp2 binds to T187-phosphorylated p27 and facilitates its polyubiquitylation by the SCF skp2 complex and subsequent proteosomal degradation.
  • H&E Hematoxylin and Eosin
  • Figure 13 shows polarity/adhesion defect in the tumor-prone Rb/p107 null retina.
  • E14 Rb/p107 null retina was stained with phallodin to mark filamentous actin (green) and DAPI (blue). The discontinuous apical actin stain and escape of cells outside the normal apical boundary is indicated with a white arrow. A blow up of the break in another Rb/107 null retina stained with phalloidin is shown below (compressed Z-stack of confocal images).
  • N-cadherin is part of the apical adherens junction that anchor filamentous actin.
  • N-cadherin stain is continuous in tumor-resistant Wild Type (WT) or Rb null retina, but aberrant breaks are detected in the tumor-prone Rb/p107 null retina (bottom picture).
  • WT tumor-resistant Wild Type
  • Rb Rb null retina
  • C Additional markers of adherens junction or regulators associated with adherens junction also reveal the phenotype.
  • D Two examples of p107 null E15 retinas in which the apical barrier is disrupted (arrows). This phenotype is never seen in the WT or Rb null retuna.
  • E Summary of genotype/phenotype correlations. Loss of Rb but not p107 causes ectopic division.
  • Loss of p107 but not Rb causes polarity/AJ defects (-20% in mixed background and 100% in C57 pure). Loss of Rb and p107 accentuates both phenotypes and is associated with a third, tumorigenesis.
  • Figure 14 shows the key polarity players.
  • the partitioning defective (PAR), Crumbs, and Scribble complexes control many polarization processes in different organisms.
  • PAR-3 and PAR-6 are PDZ domain proteins and bind the Ser/Thr kinase, atypical Pkc (aPkc) to form the PAR complex. aPkc kinase activity is essential for the function of this complex.
  • LGL tumor suppressors lethal giant larval
  • Scribble DLGI
  • the PAR3 and Crumbs-3 complexes localize predominantly to tight junctions.
  • aPkc phosphorylates Lgl to maintain the asymmetric distribution shown. From Nature Reviews Molecular Cell Biology 9, 846-849, 2008.
  • FIG. 15 shows aPkc is required for retinoblastoma in the Rb/p107 null retina.
  • the ChxIOCreiRb ⁇ plOT iPkc 4 retina is very thin and lacks tumor. Deletion of both alleles of PkcAb ⁇ ocked tumor formation and removing one allele reduced tumor frequency by 10-fold.
  • Figure 16 shows a PkcA s an essential prosurvival molecule in tumor-prone Rb/p107 null cells.
  • A P8 retinas from mice of the indicated genotypes were stained with DAPI and TUNEL or anti activated Caspase 3 (AC3). A few apoptotic cells in Wild Type (WT) and p107 null retinas represent normal pruning of retinal neurons. Rb loss induces E2f1-depedent death in a subset of differentiating neurons, which is elevated when both Rb and p107 are missing (top and bottom panels). aPkc/l removal triggered massive apoptosis of tumor prone Rb/p107 null cells at P8 (bottom panel).
  • FIG 17 shows aPkc l is required for tumor development in Rb/p107 null retinas.
  • P30 retinas from mice of the indicated genotypes were stained with DAPI, cell division markers PH3 and Ki67, and cell death markers TUNEL and activated Caspase 3.
  • Division is completed in WT retinas by P8 and therefore no dividing cell was visible at P30 in Wild Type (WT) retinas (data not shown).
  • WT Wild Type
  • DKO double knock-out
  • Figure 18 shows aPkc also promotes survival in human RB cell lines. Effect of aPKC co-knock down in WERI-RB1 (A, B, C) and Y79 (D, E, F). RB lines were co infected with two different lentiviruses carrying a resistant gene for either puromycin (*) or blasticidin (+) overnight. Coinfected cells were then selected by puromycin (7 jug/ml) and blasticidin (7 cotreatment for 7 days. aPkc t is the human counterpart of mouse aPkc i. (A and D): Knock down efficacy of coinfected cells for aPKC isoenzymes was assessed by Western Blotting after puromycin and blasticidin coselection.
  • FIG 19 shows the gold compound aurothiomalate (ATM), which inhibits aPkc interaction with the polarity regulator Par6, reaches the adult retina.
  • ATM aurothiomalate
  • ICP-AES Inductively Coupled Plasma Atomic Emission Spectroscopy
  • FIG. 21 shows ATM kills retinoblastoma cells in vitro.
  • A Effect of ATM on Y79 cell viability. Cells were treated with the indicated concentrations of ATM for 7 days and cell viability was assessed using the CellTiterGlo® luminescent kit. Average luminescence of triplicates ⁇ SD were plotted as fold change relative to day 0.
  • B, C ATM induces apoptosis. Y79 cells were treated with 50 ⁇ ATM for 5 days and were either stained live for Annexin V/PI (B), or fixed and stained for PI (cell cycle analysis), and analyzed by flow cytometry (C).
  • D Effect of ATM on caspase 3/7 activity. Cells were treated with 50 ⁇ ATM for 7 days and caspase activity was assessed using Caspase 3/7 Glo® luminescent assay kit. Average luminescence of triplicates ⁇ SD was normalized to the number of viable cells.
  • Figure 22 shows aPkc kinase activity and interaction with the polarity complex Par6 is essential for the prosurvival function of aPkc.
  • a and B Genetic loss of Pkcl reveals unique strategy to block RB initiation. Loss of Pkcl triggers massive apoptosis in P8 tumor prone retina, uncovering a novel synthetic lethal interaction.
  • aPkcl is acting through the Par complex, we transduced Rb/p107-null cells with (A) eGFP or (B) a Par6K19A dominant negative, which abolishes polarity complex function. Transfected retinas were stained with apoptosis marker activated caspase-3 (AC3).
  • AC3 apoptosis marker activated caspase-3
  • mice were treated orally every week from P28 to P56 with either vehicle or a E2f small molecule inhibitor, 6474. The mice were harvested a day after the last injection (P57).
  • Figure 24 shows a model summarizing critical molecular steps to the tumor-prone state.
  • Rb loss activates E2f1 and triggers ectopic division, but additional genetic events are required to activate Cdk2 and thus create tumor susceptibility. Sporadic mutations permit progression to cancer.
  • Rb loss delays cell cycle exit.
  • Ectopic E2f1 -dependent division stops eventually by RB-independent means.
  • Other components of the dual axes Skp2 and Cdk2
  • p107 loss lowers Rb pathway activity even further, resulting in elevated Skp2 levels and Cdk2 activity.
  • E2f activity is essentially the same as in Rb null cells. Polarity and fate are disrupted.
  • aPkc is engaged to promote survival. Most of these cells also exit the cell cycle eventually. But, this constellation of molecular events together creates an initiation network that confers the potential for sporadic tumorigenesis. This step can be reversed by modestly reducing E2f, Cdk2 or aPkc activity, or by removing Skp2. An additional sporadic event generates retinoblastoma.
  • Rb family proteins are best known as E2f inhibitors and no CKI- Cdk axis mutations have been described in human or mouse retinoblastoma. Superficially the disease thus fits a model in which enhanced E2f activity is sufficient to create tumor-prone cells.
  • Figure 1A there is extensive cross-talk between E2f and Cdk axes ( Figure 1A) (Binne et al., 2007; Buttitta et al., 2010; Buttitta et al., 2007; Ji et al., 2004; Rodier et al., 2005; Wang et al., 2010) which could lead to elevated Cdk activity when the tumor-prone state arises upon mutation of both Rb and either p107 or p130.
  • E2f was similarly high in both abnormal states, but elevated Cdk activity was specific to tumor-prone cells. Brief pharmaceutical E2f or Cdk inhibition did not perturb normal division, and only partially impaired ectopic division, yet dramatically curtailed tumorigenesis. Thus, beyond perturbing division, specific dual axes levels engender tumor susceptibility. E2f and Cdk inhibitors, untested or largely unproven as therapeutics, could be potent chemopreventive agents. Chemoprevention, the blockade of neoplasia, is particularly important for high risk cancer-prone individuals. Deducing the perturbations that underlie the tumor-prone state is critical to develop effective chemoprevention strategies.
  • Cell cycle regulation in cancer is typically viewed as normal or abnormal, but here we now define a third cell cycle state that is tumor-prone and is uniquely marked by elevated activity of E2f and Cdk cell cycle regulatory axes as well as Skp2 and aPKC.
  • exposing the cell-of-origin briefly to small molecule antagonists prevented subsequent tumorigenesis in cancer-prone mice without affecting normal division.
  • Our in vivo findings demonstrate that targeting a cell cycle state unique to the cancer cell-of-origin achieves effective and safe chemoprevention. It is proposed, therefore, that suppressing/inhibiting the members of the tumour initiation network is a feasible strategy to prevent retinoblastoma.
  • a method of preventing retinoblastoma in a subject comprising inhibiting the RB pathway.
  • the method comprises inhibiting at least one of Cdk, E2f, atypical protein kinase C, and Skp2.
  • RB pathway it is meant the entire pathway of molecular signaling that includes retinoblastoma protein (RB), and other protein/protein families in the pathway, including but not limited to Cdk, E2f, atypical protein kinase C, and Skp2.
  • Cdk, E2f, atypical protein kinase C, and Skp2 as used herein, refers individually and collectively to proteins, protein isoforms and protein families, unless indicated otherwise.
  • prevention includes any and all of primary, secondary, tertiary and quaternary prevention levels, for example, methods to avoid occurrence of disease, methods to diagnose and treat existent disease in early stages before it causes significant morbidity, methods to reduce negative impact of extant disease by restoring function and reducing disease-related complications, and methods to mitigate or avoid results of unnecessary or excessive interventions in the health system, respectively.
  • prevention includes any avoiding or mitigation of a cancerous state, including the slowing or halting of cancer progression to a more detrimental state or stage.
  • the inhibiting comprises administering to the subject an effective amount of an RB pathway inhibitor, preferably selected from the group consisting of a Cdk inhibitor, an E2f inhibitor, an atypical protein kinase C inhibitor, and a Skp2 inhibitor.
  • an RB pathway inhibitor preferably selected from the group consisting of a Cdk inhibitor, an E2f inhibitor, an atypical protein kinase C inhibitor, and a Skp2 inhibitor.
  • an effective amount is meant a nontoxic but sufficient amount of an active compound or composition to provide the desired therapeutic or preventative effect.
  • dose of a Cdk inhibitor, an E2f inhibitor, an atypical protein kinase C inhibitor, or a Skp2 inhibitor compound effective to relieve, ameliorate, or prevent symptoms of the condition or disease being treated, e.g. cancer, such as retinoblastoma.
  • Various methods of suppressing proteins/protein families are generally known in the art (Oncogene suppression by small interfering RNAs. Heidenrich O., Curr Pharm Biotechnol. 2004, 5(4): 349-54) and also specifically with respect to:
  • E2f Resveratrol induces apoptosis in breast cancer cells by E2F1 -mediated up-regulation of ASPP1 Shi et al. Oncology reports 25: 1713-1719, 2011 ; Lentivirus-mediated RNA interference of E2F-1 suppresses Tca8113 cell proliferation. Yuan et al.. Molecular Medicine Reports. 5: 420-426, 2012; Lentivirus-mediated RNA interference targeting E2F-1 inhibits human gastric cancer MGC-803 cell growth in vivo. Wang et al. Experimental and Molecular Medicine, 43: 638-645, 2011; Selective inhibition of rRNA transcription downregulates E2F-1 : a new p53-independent mechanism linking cell growth to cell proliferation. Donati et al. Journal of Cell Science 124, 3017-3028, 2011.);
  • Cdk Downlink RNA
  • aPKC atypical (aPKC; aPKCf and aPKC/ ) PKCs (Coordination of glioblastoma cell motility by PKC/.
  • the subject has cells with mutated Rb or cyclin-dependent kinase inhibitor, preferably p27, and the RB pathway inhibitor is a Cdk inhibitor or an E2f inhibitor.
  • the Cdk inhibitor inhibits at least one of Cdk1 and Cdk2. In some embodiments, the Cdk inhibitor inhibits both Cdk1 and Cdk2. In some embodiments, the E2f inhibitor inhibits at least one of E2f 1 , E2f2 and E2f3. In some embodiments, the E2f inhibitor inhibits E2f 1.
  • the E2f inhibitor is selected from the group consisting of 4- hydroxynonenal, E2f aptamer, Eugenol, thymoquinone, HLM006474, and Lithium; preferably Eugenol, thymoquinone, HLM006474 and Lithium; further preferably HLM006474 and Lithium.
  • the Cdk inhibitor is selected from the group consisting of Flavopiridol, SNS-032 (derivative 4), BS-181 hydrochloride, Indirubin, PHA-703887, [Ala92]-p16 (84-103), P1446A-05, SNS-032, AZD5438, BAY80-3000, JNJ7706621 , SCH 727965, CDKI-71 , Bohemine, RO-3306, (R)-DRF053 dihydrochloride, SU 9516, WHI-P180, Hydrochloride, CVT-313, GW8510, (R)-DRF053, NU6140, Cdc2-Like Kinase Inhibitor, TG003, Hymenidin, Iso-olomoucine, lndirubin-3'-monoxime-5- sulphonic Acid, Butyrolactone I, lndirubin-3'-monoxime, Purvalanol A,
  • the subject has cells with mutated Rb or p107, and the RB pathway inhibitor is an atypical protein kinase C inhibitor or a Skp2 inhibitor.
  • the atypical protein kinase C inhibitor is aurothiomalate or ICA- 1.
  • an RB pathway inhibitor for the prevention of retinoblastoma in a subject.
  • a use of an RB pathway inhibitor in the preparation of a medicament for the prevention of retinoblastoma in a subject is provided.
  • an RB pathway inhibitor for use in the prevention of retinoblastoma in a subject.
  • a method of determining the likelihood of a population of cells to be cancer-prone comprising: determining the level of E2f and Cdk activity in the population of cells, wherein a high level of both E2f and Cdk activity in the population compared to a control population is indicative of the population being cancer-prone.
  • a method of determining the likelihood of a population of cells to be cancer-prone comprising: determining the level of atypical protein kinase C activity in the population of cells, wherein a high or abnormal level of atypical protein kinase C in the population compared to a control population is indicative of the population being cancer-prone.
  • a method of determining the likelihood of a population of cells to be cancer-prone comprising: determining the level of Skp2 activity in the population of cells, wherein a high or abnormal level of both Skp2 activity in the population compared to a control population is indicative of the population being cancer-prone.
  • the level of protein activity is preferably determined using one of Western blot, realtime PCR or RT PCR and kinase assay.
  • the methods further comprise preventing retinoblastoma by administration of a Cdk inhibitor, an E2f inhibitor, an atypical protein kinase C inhibitor or a Skp2 inhibitor.
  • mice Mouse strains and genotyping. Mice were treated according to institutional and national guidelines. aCre mice, Rb mice, p107 ⁇ ⁇ mice, p2T' ⁇ mice, and p27° k ⁇ /Ck ⁇ mice, were maintained on a mixed background. p107 and E2f1 are in close proximity on chromosome 2, thus E2f1 + ⁇ ;p107 * mice were interbred and after analysis of > 150 pups one was identified in which a crossover had occurred to generate an E2fT;p10T chromosome. Mice of different genotypes were compared within the same litter and across a minimum of three litters. We have not noted any phenotypic differences in separate litters. Genotyping was performed as before (Besson et al., 2007; Chen et al., 2004). Histology and immunofluorescence.
  • BrdU labelling, fixation and immunostaining were essentially as described before (Chen et a!., 2007; Chen et al., 2009).
  • antigen retrieval was performed by boiling sections in citric acid solution (H-3300, Vector Lab Inc.) for 15 min.
  • RNA extraction RNA extraction, reverse transcription and PCR.
  • RT and qPCR for E2f targets were run in duplicate on at least three separate biological samples as described (Chen et al., 2009). Values obtained for test RNAs were normalized to Hprt mRNA levels. Western blots.
  • Mouse retinas were homogenized by passing them through a 30-gauge needle (BD) 5- 10 times in lysis buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. Blots were blocked and probed as described (Chen et al., 2007). Blots were scanned using ODYSSEY Infrared Imaging System (LI-COR Biosciences).
  • Agarose beads were removed by centrifugation at approximately 1 ,000xg for 5 min at 4 °C. Supernatant containing 250 ⁇ g total cellular protein was incubated with 1 /vg of primary antibody at 4°C for 2hrs. 20 ⁇ of resuspended volume of Protein A/G Plus-Agarose was added and incubated at 4 °C on a rocker platform for 1hr to overnight to pull down primary antibodies. Immunoprecipitates were collected by centrifugation at approximately 1 ,000xg for 5 min at 4 °C. Pellets were washed three times with PBS and once with 1x kinase buffer (Cell Signaling).
  • R547 and 6474 were synthesized by University Health Network, Shanghai and purity confirmed at >98% according to published methods (DePinto et al., 2006; Ma et al., 2008). Male and female mice were mixed in the early afternoon, checked the following morning and dams with vaginal plugs were considered to be 0.5 days post-coitus (E0.5). After twelve days, pregnant dams were treated with either vehicle (2.5% v/v DMSO (5% for 20mg/kg dose), 28% w/v 2-hydroxypropyhff-cyclodextrin, 10% v/v PEG400 in distilled water), or R547 (5-20 mg/kg;) or 6474 (100 mg/kg) daily intraperitoneally (I. P.) until birth. At P0, half the litter was harvested for assessment of ectopic division and the other half remained until P45 for tumor assessment.
  • vehicle 2.5% v/v DMSO (5% for 20mg/kg dose)
  • Example 2 CKI Activity Suppresses Mouse Retinoblastoma
  • Current mouse knockout models of retinoblastoma require the deletion of Rb plus either p107 or p130 (Chen et al., 2004; Dannenberg et al., 2004; MacPherson et al., 2004; Robanus-Maandag et al., 1998; Zhang et al., 2004).
  • Rb loss alone is sufficient to render cells tumor-prone
  • the mouse retina has extra Rb-like activity.
  • the additional Rb activity embodied in p107 or p130 could simply protect the mouse retina through added repression of E2f targets.
  • aCre;Rb f/ f ;p2T / ⁇ mice developed retinoblastoma with 100% penetrance ( Figure 1B and Table 1), double that of aCre;Rb f/f ;p107 / ⁇ mice and similar to aCre Rb ;p130 ⁇ ' ⁇ mice (Chen et al., 2004; MacPherson et al., 2004).
  • Rb/p27 DKO tumor cells expressed the amacrine cell marker ⁇ 2 ⁇ , as well as markers found in this and other cells such as Pax6, and Proxl, but lacked markers for other cell types ( Figure 2 and Figure 8).
  • P30 tumor cells were positive for markers that label all cell cycle phases (Ki67), M-phase (phosphohistone H3; PH3), or S-phase (BrdU).
  • p27 is a potent tumor suppressor suggesting that low CKI activity in the human retina may contribute to the sensitivity of this tissue to RB loss.
  • all mouse knockout models of retinoblastoma require deletion of Rb plus either p107 or p130 (Chen et al., 2004; Dannenberg et al., 2004; MacPherson et al., 2004; Robanus-Maandag et al., 1998; Zhang et al., 2004).
  • Rb loss alone is sufficient to render cells tumor-prone, the mouse retina has extra Rb-like activity.
  • P30 tumor cells were positive for markers that label all cell cycle phases (Ki67), M-phase (phosphohistone H3; PH3), or S-phase (Brdll), whereas contaminating glutamine synthase (GS)-labelled Muller glia were quiescent ( Figure 2B-2D and Figure 8).
  • p27 is a potent tumor suppressor.
  • CKI Cdk inhibitor
  • LOH loss of heterozygosity.
  • p27 binds and regulates proteins other than Cyclin/Cdk2 (Baldassarre et al., 2005; Besson et al., 2004; Nguyen et al., 2006).
  • Cyclin/Cdk2 Cyclin/Cdk2
  • P 27° K' CK' animals exhibit retinal dysplasia (Besson et al., 2007), which we confirmed, but they never developed retinoblastoma (data not shown). Furthermore, of the 28 eyes from aCre;Rb ;p2 /CK ⁇ animals only three had tumors, and strikingly all showed loss of heterozygosity (LOH) (Table 1 and Figure 9). Thus, p27 CK" is not a dominant oncogene in either in normal or Rb ⁇ ' retina, contrasting lung where it causes tumors (Besson et al., 2007). Collectively, our results demonstrate that retinoblastoma requires loss of p27 CKI activity.
  • p27 is the major CKI tumor suppressor in the Rb ⁇ ' ⁇ retina, and when it is missing (p27 "A ), unable to bind Cdk2 (p27 CKVCK ), or reduced following loss of p107 (piOT " ), p21 is induced, but at insufficient levels to compensate for p27.
  • E2f1 heterozygosity did not affect progenitor division, but specifically reduced ectopic division in Rb/p107-def ⁇ c ⁇ ent cells ( Figure 5B and 5C).
  • a therapeutic window of E2f activity exists that can be exploited to prevent abnormal pre-cancerous events without perturbing normal division.
  • Cdk1 can functionally substitute for Cdk2 in vivo (Santamaria et al., 2007), thus we exploited a pharmaceutical approach to inhibit both and to test a novel chemoprevention strategy.
  • Newborn neurons that survive Rblp107 loss divide ectopically, but the vast majority (millions) of neurons escape tumorigenesis by eventually exiting the cell cycle (Chen et al., 2004).
  • aCre,Rb P2T 1' males were bred to aCre;Rb f/f ;p27 +/ ⁇ females and pregnant dams received daily intraperitonial injections of vehicle or R547 (20 mg/kg) from embryonic day 12.5 (E12.5) to parturition and tumors were assessed at P45.
  • R547 20 mg/kg
  • Example 7 ATM kills tumor-prone, but not tumor-resistant retinal cells
  • the gold compound aurothiomalate (ATM) was recently identified in a screen for molecules that inhibit interaction between aPkc and the polarity regulator Par6 (Erdogan et al., 2006).
  • To test ATM levels we used Inductively Coupled Plasma Atomic Emission Spectrometry (ICP AES), as described elsewhere (Xiao-quan et al., 1987) and showed significant amounts of ATM in both serum and the retina after intraperitoneal delivery to adult mice (Figure 19).
  • ICP AES Inductively Coupled Plasma Atomic Emission Spectrometry
  • Par6-aPk ⁇ _U complex is critical for survival of tumor-prone or transformed but not normal cells, making it an ideal therapeutic target.
  • aPkc is likely pivotal for the initiation and/or sustenance of RB-pathway tumors and thus are ideal targets to prevent these cancers.
  • ATM is not a potent drug as ⁇ amounts are required, but its prior use in humans is an important advantage.
  • a new aPkc/i inhibitor ICA-1 which is ⁇ 1000x more potent than ATM has been previously described (Pillai et al., 2011).
  • aPkc inhibitor ATM specifically kills tumor-prone explanted retinal cells and retinoblastoma cell lines in vitro, suggesting that in vivo studies and examination of more potent aPkc antagonists would yield similar conclusions.
  • mice were treated orally every week from P28 to P56 with either vehicle or the E2f inhibitor, 6474. The mice were harvested a day after the last injection (P57). We show that 6474 reduces tumor volume by -14-fold, thus providing evidence that inhibiting E2f halts nascent retinoblastoma growth in vivo.
  • Rb loss delays cell cycle exit. Ectopic E2f1 -dependent division stops eventually by RB- independent means. Other components of the dual axes (Skp2 and Cdk2) are also modestly elevated. These cells never form tumors. p107 loss lowers Rb pathway activity even further, resulting in elevated Skp2 levels and Cdk2 activity. E2f activity is essentially the same as in Rb null cells. Polarity and fate are disrupted. aPkc is engaged to promote survival. Most of these cells also exit the cell cycle eventually. But, this constellation of molecular events together create an initiation network that confers the potential for sporadic tumorigenesis.
  • This step can be reversed by modestly reducing E2f, Cdk2 or aPkc activity, or by removing Skp2.
  • An additional sporadic event generates retinoblastoma.
  • cell cycle activity is loosely defined as normal or abnormal, with the latter being associated with cancer initiation.
  • distinct quanta of E2f and Cdk dual axes activity are not limited to binary states, but that beyond deregulated proliferation, core cell cycle components engage at another previously unrecognized level to create the tumor-prone state ( Figure 6F, G).
  • WT retinal progenitors had the lowest dual axes activity, while ectopically dividing Rb null cells exhibited elevated E2f activity that was required for abnormal division, but was insufficient to create cancer susceptibility (this work and (Chen et al., 2007)).
  • E2f and Cdk activity are specific characteristics of tumor-prone retinal cells. It is well known that these factors are deregulated in multiple human cancers, but our data are the first to show that discrete quanta of E2f and Cdk create dual axes that distinguish three quantal states: normal division, ectopic division and an unexpected tumor-prone state. The surprising finding that the latter two states can be molecularly separated on the basis of E2f and Cdk activity led us to the hypothesis that lowering either axis might prevent tumor initiation but not affect normal division.
  • E2f-responsive reporter vectors show comparable activity in Rb or Rblp107-nu ⁇ MEFs (Classon et al., 2000).
  • Rb but not p107/p130, inhibits E2f target expression during senescence (Chicas et al., 2010) again mimicking our findings in terminally differentiating retinal neurons.
  • p107 cannot affect E2f targets in some /3 ⁇ 4>-null contexts because it is not recruited to these genes (Chicas et al., 2010), it is redundant with p130 (Hurford et al., 1997) there is feedback inhibition of E2f by Cdk2-mediated phosphorylation (Xu et al., 1994; Dynlacht et al., 1994) and/or it is already sequestered in other complexes (Lee et al., 2002).
  • Cdk2 is an important cell cycle regulator that fires DNA replication origins.
  • Skp2 is required for f?£)-initiated mouse pituitary but not thymic tumors (Wang et al., 2010).
  • this kinase is also required for lung tumors initiated by K-ras activation (Regala et al., 2009).
  • Traditional mouse models of RB delete Rb1 and its relative, p107.
  • Skp2 binds to T187- phosphorylated p27 and facilitates its polyubiquitylation by the SCF skp2 complex and subsequent proteosomal degradation.
  • p27 T187A a knockin strain expressing a Skp2 resistant version of p27
  • oCre mouse knockout model of retinoblastoma
  • mice pituitary shows the same sensitivity to Rb loss as the human retina (Jacks et al., 1992) and we propose that the vulnerability in both these mammalian tissues reflects an unusually weak inhibitory buffer between the E2f and Cdk axes.
  • Skp2 is essential for the development of pituitary tumors in the Rb +/ ⁇ mouse, and for the growth of human retinoblastoma cell lines (Wang et al., 2010).
  • Skp2 is required for the development of retinoblastoma in the Rb/p107 null mouse retina.
  • aPkc is a prevention target for retinoblastoma.
  • Aurothiomalate (ATM) formerly used to treat arthritis, inhibits aPkc-Par6 binding13, and notably this drug also caused synthetic lethality specifically in tumor-prone but not tumor-resistant retina.
  • Cdk2 is dispensable for tumorigenesis in p27, p21 or p53-null mice (Padmakumar et al., 2009; Martin et al., 2005; Tetsu et al., 2003), it acts redundantly with Cdk1 (Santamaria et al.,
  • CDK inhibitors p18(INK4c) and p27(Kip1) mediate two separate pathways to collaboratively suppress pituitary tumorigenesis. Genes Dev 12: 2899- 2911.
  • p27kip1 independently promotes neuronal differentiation and migration in the cerebral cortex. Genes Dev 20: 1511-1524. Old, J. B., Kratzat, S., Hoellein, A., Graf, S., Nilsson, J. A., Nilsson, L., Nakayama, K. I., Peschel, C, Cleveland, J. L., and Keller, U. B. (2010).
  • Skp2 directs Myc-mediated suppression of p27Kip1 yet has modest effects on Myc-driven lymphomagenesis.
  • p107 is a suppressor of retinoblastoma development in pRb-deficient mice. Genes Dev 12: 1599-1609.
  • Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1 by phosphorylation. Mol Cell Biol 14: 8420-8431.

Abstract

L'invention concerne un procédé de prévention du rétinoblastome chez un sujet prédisposé au cancer au moyen d'inhibiteurs de la voie de RB. Des inhibiteurs préférés sont les inhibiteurs de Cdk, les inhibiteurs d'E2f, les inhibiteurs de protéines kinases C atypiques et les inhibiteurs de Skp2. Des procédés de détermination de la probabilité qu'une population de cellules soit prédisposée à la cancérisation par la mesure des niveaux d'activité de Cdk et d'E2f, d'aPKC et de SKp2 sont également décrits.
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