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WO2006037984A2 - Traitement du cancer - Google Patents

Traitement du cancer Download PDF

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Publication number
WO2006037984A2
WO2006037984A2 PCT/GB2005/003798 GB2005003798W WO2006037984A2 WO 2006037984 A2 WO2006037984 A2 WO 2006037984A2 GB 2005003798 W GB2005003798 W GB 2005003798W WO 2006037984 A2 WO2006037984 A2 WO 2006037984A2
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cell
bci
cancer
intracellular
pretumorigenic
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WO2006037984A3 (fr
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Denis Alexander
Rui Zhao
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Babraham Institute
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Babraham Institute
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Priority claimed from GB0422225A external-priority patent/GB0422225D0/en
Priority claimed from GB0507785A external-priority patent/GB0507785D0/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure

Definitions

  • This invention relates to prevention, treatment, or amelioration of cancer, particularly cancer that is resistant to genotoxic treatment.
  • Intracellular pH has an important role in the maintenance of normal cell function, and so has to be controlled within a narrow range. This is done largely through the activity of transporters located at the plasma membrane. Disturbances of pH homeostasis, particularly extracellular acidification and intracellular alkalinization, are known to be a common feature of cancer (Lagadic-Gossmann et al, Cell Death and Differentiation, 2004, 11: 953-961). It has been proposed that induction of intracellular acidification might be used to treat some types of cancer (Izumi et al, Cancer Treat. Rev. 2003, 29: 541-549), and enhancement of chemotherapy by extracellular alkalinization has been reported (Raghunand et al, British Journal of Cancer, 1999, 80(7): 1005-1011).
  • Zhao et al. (Cancer Cell, 2004, 5: 37-49) have generated a transgenic mouse model of T cell lymphoma in which an oncogene inhibits DNA repair and prevents the subsequent elimination of damaged cells by 'suppressing DNA-damage induced Bcl-xL deamidation, thereby preserving its anti-apoptqtic function.
  • a method of preventing, treating, or ameliorating cancer which comprises increasing intracellular pH of a pretumorigenic or tumour cell in a subject in need of such prevention, treatment or amelioration.
  • intracellular pH refers to cytosolic pH, not the pH of organelles within the cytoplasm.
  • pretumorigenic cell is used herein to include cells that are believed to be at increased risk of becoming or giving rise to tumour cells. This may be, for example, because they are known to express a particular antigen associated with increased risk of tumorigenesis, or because the subject has a family history of cancer in tissue that comprises cells of the same type as the pretumorigenic cells, or because the subject is known to carry a chromosomal abnormality even though clinical symptoms of cancer are absent.
  • tumour cell death which comprises increasing intracellular pH of a tumour cell.
  • Intracellular pH of tumour cells is comparable with normal cells. Intracellular pH of tumour tissues ranges from approximately 7.1 to 7.3, whereas in normal tissues intracellular pH ranges from 7.0 to 7.2 4 .
  • intracellular pH of the pretumorigenic or tumour cell is preferably increased to at least pH 7.5, more preferably at least pH 7.6, most preferably at least pH 8.0.
  • intracellular pH of the cell is increased for at least 12 hours, preferably 24 to 48 hours.
  • Intracellular pH of the pretumorigenic or tumour cell may be increased by any suitable method other than by causing DNA damage to the cell.
  • intracellular pH of the cell is increased by promoting the activity, or increasing the amount, of the NHE-I Na + /H + exchanger on the cell.
  • the NHE family of ion exchangers includes six isoforms (NHE- l-NHE-6) that function in an electroneutral exchange of intracellular H + for extracellular Na + .
  • NHE-I is the only ubiquitously expressed isoform and is localised at the plasma membrane .
  • NHE-I activity is known to be promoted in response to growth factors (Wakabayashi S, et al. . Proc. Natl. Acad. 1992Sci. USA 89: 2424-28), hormones (Winkel GK, et al. J. Biol. Chem.1993. 268:3396-400) and osmotic stress (Grinstein S, et al.J. Biol. Chem. 1992. 267: 23823-28).
  • the following agents are known to promote the activity of NHE-I: serotonin (Garnovskaya MN, et al. Biochemistry 2003, 42: 7178-87; Mukhin YV, et al. J. Biol. Chem.
  • NHE-I expression on the cell may be increased.
  • intracellular pH of the cell may be increased by promoting the activity, or increasing the amount, of a sodium/potassium ion pump on the cell.
  • Agents that are known to promote the activity of the sodium/potassium ion pump include corticosteroids, norepinephrine, insulin and insulin-like peptide hormones (Therien AG, Am. J. Physiol. Cell Physiol. 2000, 279: C541-566).
  • expression of the sodium/potassium ion pump on the cell may be increased.
  • an antibody that binds to NHE-I or the sodium/potassium ion pump is used to promote the activity of NHE-I or the sodium/potassium ion pump.
  • the antibody is a multivalent (i.e. at least bivalent) antibody capable of binding to NHE-I or the sodium/potassium ion pump to promote the activity of NHE-I or the sodium/potassium ion pump, and simultaneously to a tumour-specific antigen.
  • Such antibodies are expected to specifically target tumour cells.
  • the antibody binds to at least two tumour-specific antigens and NHE-I or the sodium/potassium ion pump.
  • Such antibodies are expected to have higher specificity for tumour cells compared to antibodies that bind to a single tumour-specific antigen.
  • binding of the antibody to NHE-I or the sodium/potassium ion pump cross-links molecules of NHE-I or the sodium/potassium ion pump .
  • the subject is a human subject.
  • the antibody is preferably a humanised antibody to minimise any rejection of the antibody by the subject.
  • antibody is used herein to include a fragment or derivative of an antibody.
  • intracellular pH of the pretumorigenic or tumour cell is increased by increasing the extracellular sodium ion concentration of the pretumorigenic or tumour cell in order to stimulate extrusion of hydrogen ions from the pretumorigenic or tumour cell by NHE-I. This could be achieved, for example, by contacting the prerumorigenic or tumour cell with salt.
  • the pretumorigenic cell or tumour cell is also subjected to a genotoxic treatment.
  • a genotoxic treatment Increasing intracellular pH of the pretumorigenic or tumour cell other than by causing DNA damage to the cell is expected to increase the susceptibility of the cell to genotoxic treatment. It will be appreciated that this may be particularly useful against cancers that are resistant to genotoxic treatment. Hepatocellular carcinoma is known to be highly resistant to genotoxic treatment. Other types of cancer that may be resistant to genotoxic treatment include breast, central nervous system, colon, lung, lymphoid, melanoma, ovarian, prostate, renal, bladder, brain, and oesophageal cancer.
  • Cancers where there is 52% or less age standardised relative survival within 5 years from diagnosis are considered to be cancers which are resistant to genotoxic treatment.
  • such cancers include non-Hodgkins lymphoma (47%), rectal (47%), colon (47%), kidney (45%), leukaemia (36%), multiple myeloma (23%), stomach (13%), brain (12%), oesophageal (7%), lung (6%), pancreas (2%).
  • cancers include non- Hodgkins lymphoma (52%), rectal (51%), colon (48%), kidney (44%), ovarian (36%), leukaemia (35%), multiple myeloma (23%), stomach (15%), brain (15%), oesophageal (8%), lung (6%), pancreas (3%).
  • the percentages given are from CancerStats Monograph 2004, Chapter 2, Figure 1, page 7, Cancer Research UK.
  • Genotoxic treatments are well known to those of ordinary skill in the art. Any suitable- genotoxic treatment may be used.
  • the cell may be subjected to radiotherapy and/or the cell may be contacted with a chemotherapeutic agent. Examples of chemotherapeutic agents are included in Table 2 of Amundson et al (reference 24 below).
  • the chemotherapeutic agent is an agent that preferentially targets to cells with an intracellular pH corresponding to the intracellular pH to which the pretumorigenic or tumour cell is increased, i.e. the agent targets to cells which have the increased intracellular pH in preference to normal cells with a lower intracellular pH.
  • the preference of the agent for cells with the increased intracellular pH may be 10 times or 100 times greater than for normal cells.
  • increasing the intracellular pH of the pretumorigenic or tumour cell is expected to help target the chemotherapeutic agent to the pretumorigenic or tumour cell, thereby increasing the efficacy of methods of the invention, and reducing side effects.
  • Figure 7 shows that the proportion of deamidated BCI-X L expressed (as a percentage of the total BCI-X L expressed) in different tumours varies between the different tumours. It is believed that tumours with a relatively high proportion of deamidated BcI- X L are more likely to be susceptible to genotoxic treatment, whereas those tumours with a lower proportion of deamidated BCI-X L may be more likely to be resistant to genotoxic treatment. In particular it is believed that tumours that normally fail to further deamidate BCI-X L following genotoxic attack will be killed more effectively by enforced intracellular alkalinization.
  • Methods of the invention are expected to be particularly advantageous against cancers in which a tumour of the cancer express non-deamidated BCI-X L at a level which is at least 50%, preferably at least 60%, 70%, or 80%, and more preferably at least 90%, of the total BCI-X L expressed by the tumour.
  • the results shown in Figure 7 are from analysis of tumours from the following cancers: squamous cell carcinoma lymph node metastasis, papillary Adeno-Carcinoma of the ovary, neuroendocrine Carcinoma of the prostate- lymph node metastasis, B-CLL, clear cell papillary Carcinoma of the kidney- lymph node metastasis, poorly, partly neuroendocrine diff. prostate Care- lymph n.
  • lobular mammary gland carcinoma, ductal mammary gland carcinoma, PNET mucoepidermoid carcinoma- lymph node metastasis, poorly differentiated, rarely cornyfying Carcinoma of sinus pirif., paragranuloma like T-cell lymphoma, diffuse large B -cell lymphoma, mantle cell lymphoma, mammary gland Carcinoma, ductal and lobular, diffuse large B-cell lymphoma- immunoblastic, follicular lymphoma, grade 1. It is believed that methods of the invention may be effective against any of these cancers.
  • methods of the invention may be particularly effective in the prevention, treatment, or amelioration of cancer in which an oncogenic tyrosine kinase (OTK) is expressed.
  • OTK oncogenic tyrosine kinase
  • T-lineage cancers intracellular pH is increased in a pretumorigenic or tumour cell which is a T- lineage cell.
  • T-lineage cancers include leukaemias and lymphomas, such as T cell lymphoma.
  • the change in the level of deamidated BCI-X L expressed by a tumour (as a proportion of the total BCI-X L expressed by the tumour) during treatment in accordance with methods of the invention can be used to monitor the progress and likely effectiveness of the treatment. If the level of deamidated BCI-X L increases during treatment, then it can be concluded that the increase in intracellular pH of the tumour cells caused by the treatment is sufficient. If, however, the proportion of deamidated BCI-X L expressed by the tumour is not significantly increased, then the treatment should be changed to promote a further increase in the intracellular pH of the tumour cells.
  • a method of monitoring cancer treatment which comprises monitoring change in the proportion of deamidated BCI-X L relative to total BCI-X L expressed by a tumour in response to treatment.
  • a method of treating or ameliorating cancer which comprises: increasing intracellular pH of a tumour cell of a tumour in a subject in need of such treatment or amelioration; monitoring change in the proportion of deamidated BCI-X L relative to total BCI-X L expressed by the tumour in response to the treatment; and further increasing intracellular pH of the tumour cell if the proportion of deamidated BCI-X L relative to total BCI-X L expressed by the tumour is not increased in response to the treatment.
  • Intracellular pH of the tumour cell should be increased other than by causing DNA damage to the cell.
  • an agent that increases, or causes an increase in, intracellular pH of a pretumorigenic or tumour cell in the manufacture of a medicament for the prevention, treatment, or amelioration of cancer.
  • the agent should cause an increase intracellular pH of the cell other than by causing DNA damage to the cell.
  • the agent increases intracellular pH of the cell to at least pH 7.5, preferably at least pH 7.6, more preferably at least pH 8.0.
  • the agent optionally further comprises a genotoxic agent.
  • CD45 " " lck Y505F mice may be used to screen for agents that increase intracellular pH of a pretumorigenic or tumour cell.
  • a method of screening for an agent that increases intracellular pH of a pretumorigenic or tumour cell which comprises contacting a candidate agent with a pretumorigenic or tumour cell of a CD45 '/ lck Y505F mouse, and measuring intracellular pH of the cell to determine whether the intracellular pH is increased by the candidate agent.
  • the candidate agent is isolated and/or identified if intracellular pH of the cell is found to be increased by the candidate agent.
  • CD45 ' " lck Y505F mice may also be used to screen for agents that prevent or inhibit tumorigenesis either with or without genotoxic therapy.
  • a method of screening for an agent that prevents or inhibits tumorigenesis which comprises contacting a candidate agent with a pretumorigenic cell of a CD45 " " lck YS0SF mouse, and determining whether tumorigenesis is prevented or inhibited by the candidate agent.
  • the candidate agent is isolated and/or identified if tumorigenesis is prevented or inhibited.
  • a method of promoting deamidation of BCI-X L in a pretumorigenic or tumour cell which comprises increasing intracellular pH of the cell.
  • a method of prevention, treatment, or amelioration of cancer which comprises promoting BCI-X L deamidation in a pretumorigenic or tumour cell, in a subject in need of such prevention, treatment or amelioration.
  • an agent that promotes BCI-X L deamidation in a pretumorigenic or tumour cell in the manufacture of a medicament for the prevention, treatment, or amelioration of cancer.
  • Bim binds to the native (Asn-Asn) but not deamidated forms of BCI-X L - Either wild-type (C57BL/6) or pre-tumorigenic CD45 "/" Lck F505 thymocytes were exposed to 5 Gy irradiation (IR) and then maintained hi culture for the times shown, after which cells were lysed and either separated as whole cell lysates (WCL) or as Bim immunoprecipitates, followed by immunoblotting for either BCI-XL ° ⁇ for Bim. Bim migrates as 'extra-long' (EL) or 'long' (L) forms.
  • Puma binds to the native but not deamidated form of BCI-X L .
  • Either wild-type or pre-tumorigenic CD45 " " Lck F50 thymocytes were treated as in (a), and cells were lysed and subjected to immunoprecipitation with Puma, followed by blotting with either BCI-X L or Puma.
  • the asterisk indicates the light chain of Puma antibody used in immunoprecipitation.
  • Mitochondrial extracts were prepared from wild-type thymocytes maintained in culture for the times shown after exposure to irradiation prior to Bim immunoprecipitation. The immunoprecipitates and Bun depleted lysates were separated and blotted for either BCI-X L or for Bim.
  • Bim binds to native " but not to deamidated ⁇ BCI-X L .
  • the three different forms of BCI-X L (A, B and C) purified by anion-exchange column chromatography shown in (d) were incubated in wild-type thymic lysates at 4 C for 2 h and then precipitated using Nickel beads. The precipitated products were immunoblotted for Bim and BCI-X L . Quantification of the Bim-L/Bcl-X L ratios +/-SD from three independent experiments is shown in the histogram, with the Lane A ratio normalised to 1 (*).
  • Intracellular alkalinization occurs following DNA damage in wild-type but not in pre-tumorigenic CD45 "/" Lck F50S thymocytes.
  • Cells were treated with Etoposide (E) for 20 h or exposed to 5 Gy of irradiation (IR) and then maintained in culture for 2Oh.
  • Intracellular pH (pH;) was measured using SNARF either on whole thymocytes in a spectrofluorimeter ('Thymocytes', left panel) or by FACS in the gated live CD4 " CD8 ' sub-set (middle panel).
  • the right panel shows by FACS the sub-Gl peak of Propidium Iodide-stained CD4 ' CD 8 " wild-type thymocytes either untreated (U), or treated for 5h or 24h with etoposide (E5 and E24), or incubated for 5h or 24h following 5 Gy irradiation (IR5 and IR24), in Hepes-buffered media at pH 6.0, 6.5 or 7.2.
  • Wild-type thymocytes were treated in neutral (pH 7) or alkaline (pH 9) buffer at 37 0 C for 24h. Mitochondrial extracts were prepared and immunoprecipitated for Bim. The WCL, Bim immunoprecipitates and Bim-depleted lysates were separated and blotted for either BCI-X L O ⁇ Bim.
  • BCI-X L deamidation induced by DNA damage requires de novo protein synthesis.
  • Wild-type thymocytes were either treated with Etoposide for 24 h, or exposed to 5 Gy of irradiation and then maintained in culture for 24 h, with or without 0.5 ⁇ M Cycloheximide (CHX).
  • Cell lysates were processed by immunoblotting for Bcl-X L ⁇ r ⁇ -actin (loading control).
  • the NHE-I inhibitor DMA blocks DNA-damage induced alkalinisation (top left panel), BCI-X L deamidation (lower panel) and apoptosis (top right panel) in wild- type thymocytes.
  • Thymocytes were treated with Etoposide for 24 h, or exposed to 5 Gy of irradiation and then maintained in culture for 24 h, with or without 200 ⁇ M DMA. pHi was measured by FACS on live CD4 " CD8 " cells, and the sub-Gl peak was analysed by FACS on CD4 " CD8 ' cells to assess apoptosis.
  • NHE-I or empty vector were transduced into wild-type or pretumorigenic CD45 "7" Lck F505 thymocytes.
  • 72 h after the first round of infection cells were immunoblotted for NHE-I and BCI-X L .
  • NHE-I expression levels (NHE-I relative intensity) were normalised for loading using tubulin values. Deamidation were calculated as in Fig. 2c.
  • the lower left FACS histogram shows the infection efficiency for non-transfected (non), empty- vector transfected (vector) or NHE-I transfected (NHE-I) cells as percentage GFP positive cells.
  • the lower right histograms show the pHiand apoptosis (sub-Gl) values analysed on GFP negative and positive cells.
  • Figure 4. NHE-I knock-down blocks DNA-damage induced BCI-X L deamidation and apoptosis.
  • NHE-I siRNA (si 1-5), negative control and empty vector were transduced into wild- type thymocytes. Immunoblotting for NHE-I and tubulin shows si2 is the most potent siRNA inhibiting NHE-I expression, so si2 was used in subsequent experiments.
  • NHE-lsiRNA (si2) was transduced into wild-type thymocytes treated with Etoposide or irradiation prior to immunoblotting for NHE-I and BCI-X L .
  • CD45 "/” Lck F505 tumour cells were cultured in the media with the pH e as shown without monensin, treated with irradiation or Etoposide, and analysed for BCI-X L deamidation by immunoblotting. The percentage deamidation was calculated as in Fig2(d).
  • DNA-damage induced intracellular alkalinisation in wild-type thymocytes is both necessary and sufficient to explain BCI-X L deamidation, triggering apoptosis, whereas in pre-tumorigenic thymocytes and tumour cells alkalinisation is inhibited, preventing BCI-X L deamidation.
  • Alkalinisation is dependent on increased expression of the NHEl Na + /H + exchanger, an increase blocked in pre-tumorigenic thymocytes and tumour cells.
  • Our findings suggest that oncogenic suppression of DNA-damage triggered alkalinisation is critical for T cell carcinogenesis and indicate a novel approach to chemotherapy.
  • Protein deamidation has broad biological implications, ranging from changes in the specificity of antigen presentation (Moss et al., 2005), to modifications in eye lens proteins (Hanson et al., 2000), to reduced binding of CD4 to HIV gpl20 (Teshima et al., 1991), to the activation of RhoA by cytotoxic necrotizing factor (Hoffmann et al., 2004), to aging (Robinson and Robinson, 2004), to name but a few examples.
  • GIn proceeds both enzymatically and nonenzymatically in physiological systems, whereas only the nonenzymatic deamidation of internal Asn residues has been reported, involving conversion to Iso-Asp:Asp in a ratio of about 3:1, the precise ratio depending on the environment of the Asn residue (Aswad et al., 2000; Robinson and Robinson, 2004).
  • Deamidation of both GIn and Asn residues in vitro can be greatly accelerated by exposure to either acid or alkaline pH, with minima in the range pH 4 - 6.
  • BCI-XL deamidation in response to DNA damage occurs at two internal Asn residues (Asn-52 and Asn-66) causing a characteristic retardation on SDS-PAGE gels ⁇ ' 12 .
  • Initial work from the Weintraub laboratory suggested that when Asn-52 and Asn-66 are both mutated to Asp, then BCI-X L loses its ability to bind to the BH3-only pro-apoptotic protein Bim, thereby providing a putative linkage between DNA damage and apoptosis .
  • a secondary mutation was later identified which, when corrected, enabled the N52D/N66D BCI-X L to bind Bim, casting doubt on this interpretation 13 .
  • DNA- damage induced apoptosis is suppressed in pretumorigenic thymocytes, correlating with inhibition of BCI-X L deamidation, Bax conformational change and mitochondrial translocation, cytochrome c release, and the apoptotic caspase execution cascade.
  • Bcl-X L deamidation is a critical switch in oncogenic kinase- induced T-cell transformation, and suggested that BCI-X L deamidation to an Iso- Asp52/Iso-Asp66 version, rather than the mutant N52D/N66D version investigated by the Weintraub laboratory, might be the key step in disabling the anti-apoptotic functions of the protein 9 10 .
  • DNA-damage triggered deamidation in primary wild-type cells is mediated not enzymatically, but by intracellular alkalinisation caused by increased expression of the NHE-I Na + ZH + exchanger (antiport), events blocked by expression of the oncogenic tyrosine kinase.
  • enforced alkalinisation triggers BCI-X L deamidation, crippling its ability to provide protection from the pro-apoptotic consequences of DNA damage, thereby indicating possible novel approaches to cancer therapy.
  • BCI-X L deamidation in situ involves conversion of Asn-52/Asn-66 to Iso-Asp-52/Iso- Asp-66 blocking sequestration of Bim and PUMA
  • Figure Ia shows that BCI-X L , measured in whole cell lysates from either wild-type (C57BL/6) or pre-tumorigenic CD45 " " Lck F5 ° murine thymocytes, migrates as a doublet on SDS-PAGE gels, comprising the faster migrating N52/N66 protein, and the slower migrating deamidated versions (Ref. 12 and cf. Fig. Ie). Exposure of wild-type thymocytes to ⁇ -irradiation induced the characteristic loss of the lower band due to deamidation after 36h but not 6h, and this deamidation was blocked in pretumorigenic thymocytes, consistent with our previous findings 9 .
  • Figure 2a shows that the mean average pH; in wild-type thymocytes increased from 7.2 to 8.5 within 2Oh following DNA damage, consistent with previously published results 19 . Strikingly, however, this increase was markedly suppressed in the pre-tumorigenic thymocytes. Measurements of pHi in whole cell populations do not distinguish between live and dead cells, raising the possibility that increased pHi is a consequence rather than a cause of apoptosis. We therefore carried out the analysis by flow cytometry, gating out dead cells and gating on the CD4 " CD8 " thymic sub-set (Fig. 2a, middle panel).
  • FIG. 2c illustrates two critical controls. The left panel shows that when DNA-damage was induced in wild-type thymocytes, BCI-X L deamidation could be prevented by equilibrating pHj to 6.5 or lower in the presence of monensin.
  • the right panel shows that the resistance to BCI-X L deamidation observed in DNA- damaged pre-tumorigenic thymocytes could be completely overcome by increasing the pH to 8.0 or above. Since monensin itself causes some thymic apoptosis, we repeated these control experiments in the absence of monensin (Fig2e), monitoring pHj (Fig.2f) and apoptosis (Fig. 2g) in aliquots of the same cells used to assess BCI-X L deamidation.
  • Figure 2h shows that, as expected, the Ala-52/Ala-66 mutant migrates as the lower non-deamidated version of BCI-X L , whereas Asp-52/Asp-66 migrates as the more negatively charged deamidated version.
  • the apoptosis induced by enforced alkalinisation was dramatically reduced compared to cells transduced with empty vector or with the wild-type protein (Fig 2h, right panel), demonstrating that BCI-X L in a version able to sequester BH3-only proteins protects thymocytes from an enforced increase in pHj.
  • DNA damage induced alkalinisation, BCI-X L deamidation and apoptosis are mediated by increased NBDE-I antiport expression.
  • Figure 3 a shows that de novo protein synthesis is essential for BCI-X L deamidation following DNA-damage in wild-type thymocytes. Since the NHEl Na/H antiport is a well-established regulator of pHi 20 we measured its expression in wild-type thymocytes after DNA-damage and found that the NHEl level increased 2.5-fold within 5h, whereas this increase was completely suppressed in pre- tumourigenic thymocytes (Fig. 3b).
  • FIG. 3e illustrates (upper panel), a 2-3-fold expression of NHE-I in pre-tumorigenic thymocytes, without DNA damage, restored BCI-X L deamidation to a level comparable with that observed in a retrovirally transduced wild-type control, thereby bypassing the OTK-mediated block in these events.
  • Over-expression of NHE-I increased both pHj and apoptosis to comparable levels in both pre-tumorigenic and wild-type thymocytes (Fig. 3e lower panels).
  • Figure 4a shows that 3/5 different RNAi sequences achieved substantial depletion of NHE-I, whereas no depletion was observed using the negative control sequence, and Si2 RNAi was chosen for further studies as it routinely achieved an 80% reduction or more in NHE-I expression.
  • NHE-I knock-down almost completely blocked the actions of DNA damage in causing BCI-X L deamidation (Fig. 4b), intracellular alkalinisation (Fig. 4c) or apoptosis (Fig. 4d).
  • Fig. 4b shows that BCI-X L deamidation
  • Fig. 4c intracellular alkalinisation
  • Fig. 4d apoptosis
  • FIG. 4d illustrates, double- staining for Annexin-V and propidium iodide (PI) followed by FACS analysis revealed a major increase in Annexin-V + Pr (apoptotic) cells following transduction with the negative control RNAi followed by either ⁇ -irradiation or treatment with etoposide, whereas there was no increase in apoptotic cells above base-line in the cells depleted of NHE-I. Comparable results were obtained by measuring the sub-Gl (fragmented DNA) peak by FACS (data not shown).
  • NHE-I antiport in addition to regulation of its expression, post-translational modification of the NHE-I antiport might also be involved in mediating the DNA damage response.
  • a number of serine kinases have been shown to regulate NHE-I phosphorylation and activity 21l2Z
  • Enforced alkalinisation causes increased BCI-X L deamidation and apoptosis in murine tumor cells.
  • Figure 5a shows that BCI-X L deamidation following DNA-damage was suppressed in primary tumour cells to the same extent as in pre-tumorigenic thymocytes 24h after inducing DNA damage, although after 48 h the inhibition of deamidation was somewhat less (68.1+5.2% inhibition in tumour cells cfd to 96.2+3.8% in pre-tumourigenic thymocytes, data not shown).
  • alkalinisation Fig. 5b upper panel
  • apoptosis Fig. 5b, lower panel
  • increased NHE-I expression Fig. 5c
  • BCI-X L expression levels in human tumour cells correlate with resistance to apoptosis induced by genotoxic reagents 10 ' 24 and that BCI-X L deamidation is inhibited in hepatocellular carcinomas which are highly resistant to genotoxic treatments .
  • a wide range of human tumours express high levels of native non-deamidated BCI-X L (Figure 7).
  • Our results suggest that this resistance could be overcome by increasing the tumour pHi to 7.5 or above in order to induce BCI-X L deamidation.
  • Targeted stimulation of the NHE-I antiport on tumour cells, pharmaceutical induction of NHE-I expression, or increasing tumour pHi by manipulation of other acid/base regulatory mechanisms, might therefore significantly increase chemotherapeutic efficacy.
  • Enforced alkalinisation causes increased BCI-X L deamidation and apoptosis in human cancer cells.
  • Chronic lymphocytic leukaemia CLL is the most common adult haematological malignancy in the Western world and, like many cancers, is characterised by the development of drug resistance.
  • CLL Chronic lymphocytic leukaemia
  • Figs. 5 primary murine thymocytes
  • the B-CLL cells behaved more like wild- type thymocytes in that DNA damage at physiological pH caused a mean increase of pHi 0.25 unit, 20% more BCI-X L deamidation and 17% more cells undergoing apoptosis (Fig. 6b), compared to thymocyte values of 0.45 pHi unit, 42% and 37%, respectively.
  • B-CLL cells behaved in a comparable way to both murine thymocytes and tumour cells.
  • NHE-I expression in response to DNA damage was investigated in a further 6 B- CLL patients.
  • Figure 6b shows by immunoblotting (right panel) that there was some variation between patients, but that in all cases (left panel) etoposide caused increased NHE-I expression by 3h, achieving optimal values by 6-9h ranging from 1.7-2.6-fold over basal. These increases correlate with the observed increases in BCI-X L deamidation and apoptosis in patients' cells (Fig. 6a) and are comparable with those observed in wild- type thymocytes (Fig. 5c).
  • CML chronic myelogenous leukaemia
  • p.vera polycythaemia vera
  • OTK oncogenic tyrosine kinases
  • mice were bred and housed in specific pathogen free conditions in the animal facility at The Babraham Institute, Cambridge, UK.
  • the p56 Lck"F505 (PLGF-A) transgenic mice 26 and the CD45 " ' " , CD45- / Xck FS05 , Rag 'Xck 17505 , and Rag “/" CD45- / Xck F505 mice have been previously described 8
  • Cycloheximide (CHX), 5-(N,N'-dimethyl)-amiloride (DMA), propidium iodide, monensin, nigericin, goat-anti-rat Ig- Agarose, were from Sigma (St.
  • Freshly isolated thymocytes were irradiated with 10 Gy using a caesium source or treated with etoposide at a concentration of 25 ⁇ M for the times indicated.
  • rat Bim antibody Oncogene, San Diego was coated to goat-anti-rat Ig-Agarose; rabbit Puma antibody was coated to protein A-Sepharose; mouse NHE-I antibody was coated to protein G-Sepharose. Lysates were pre-cleared with the appropriate agarose. Quantification of immunoblots was carried out using a phosphorimager (Fuji FLA3000).
  • BCI-X L ( ⁇ BCI-X L - 6 x His) was expressed in E. coli and purified using Co 2+ chelation beads so that rapid elution could be performed at pH 7.0 to prevent deamidation.
  • mass spectrometric analysis was carried out by desalting aliquots (l ⁇ l) of each Peak by solid- phase microextraction on C4 Zip Tips (Millipore) and the proteins eluted with 0.1% formic acid/50% aqueous acetonitrile (l ⁇ l) directly into a nanospray tip (Protana Engineering).
  • the nanospray tip was inserted into a nanoelectrospray ion source (Protana Engineering) attached to a quadupole-time-of-flight mass spectrometer (Qstar Pulsar i, Applied Biosystems-MDS Sciex) and full scan TOF spectra were acquired at an ionization potential of 900V for 5 min over the m/z range 500-2000 atomic mass units.
  • the mass spectrometric data were averaged and deconvoluted using the Bayesian Protein Reconstruct function in Bio Analyst software (Applied Biosystems).
  • each ⁇ BCI-X L species was added to C57BL/6 thymocyte lysates for 2h at 4 0 C at pH 7.2, and Ni 2+ beads were used to precipitate the rBcl-XL.and complexed Bim.

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Abstract

L'invention concerne des méthodes de prévention, de traitement ou d'atténuation du cancer, en particulier du cancer résistant à un traitement génotoxique. Ces méthodes consistent à augmenter le pH intracellulaire d'une cellule tumorale ou prétumorigène autrement qu'en endommageant l'ADN de la cellule.
PCT/GB2005/003798 2004-10-01 2005-09-30 Traitement du cancer Ceased WO2006037984A2 (fr)

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GB0421734.5 2004-10-01
GB0421734A GB0421734D0 (en) 2004-10-01 2004-10-01 Treatment of cancer
GB0422225A GB0422225D0 (en) 2004-10-07 2004-10-07 Treatment of cancer
GB0422225.3 2004-10-07
GB0507785A GB0507785D0 (en) 2005-04-18 2005-04-18 Treatment of cancer
GB0507785.4 2005-04-18

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