WO2011029946A1 - Produit protéolytique d'histone h2a en tant que marqueur pour le cancer - Google Patents

Produit protéolytique d'histone h2a en tant que marqueur pour le cancer Download PDF

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Publication number
WO2011029946A1
WO2011029946A1 PCT/EP2010/063425 EP2010063425W WO2011029946A1 WO 2011029946 A1 WO2011029946 A1 WO 2011029946A1 EP 2010063425 W EP2010063425 W EP 2010063425W WO 2011029946 A1 WO2011029946 A1 WO 2011029946A1
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histone
peptide
cells
present
cll
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PCT/EP2010/063425
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English (en)
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Maarten Dhaenens
Dieter Deforce
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Universiteit Gent
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Definitions

  • the present invention relates to proteolytic products of histone H2A.
  • the present invention discloses a peptide having the amino acid sequence VTIAQGGVLPNIQAV which can be used to determine cleavage of histone H2A. More in particular, the present invention relates to the latter peptide as a marker to diagnose or to determine prognosis of cancer such as chronic lymphocytic leukemia.
  • Histone H2A is one of the 5 histones which are present in nuclei of eukaryotic cells. Histones form nucleosomes and are important modulators of several important cellular processes such as transcription, translation and DNA-repair mechanisms (Wolffe and Pruss, 1996; Zhang et al. 2003). Histones such as H2A undergo post-translational modifications which are important to fulfill their biological functions. For example, a secondary modification can inhibit transcription of a specific DNA sequence whereas removal of that modification results in transcription of this DNA sequence (Minami et al., 2006).
  • Histone H2A more in particular histone H2A type ID (UniProtKB/Swiss-Prot P20671, H2A1D_HUMAN at www.uniprot.org) consists of 129 amino acids (without its initiator methionine), weighs about 14kDa and at least 3 post-translational modifications have been described: a phosphorylation on Seri, an acetylation on Lyss and an ubiquitination on Lysn 9 (Peterson and Laniel, 2004). The latter ubiquitination alters the molecular weight of H2A1D from 14kDa to 23kDa.
  • H2A isoforms such as type HI A B/C/D/H/J, type H2 B/C and H3, and variants of H2A such as macroH2A of H2A.Y (40, 1 kDa), H2A.Z (13, 5 kDa), H2A.x (15.1 kDa) and H2A-Bbd (12.7 kDa) are known (Chakravarthy et al, 2004).
  • Chronic Lymphocytic Leukemia is a very heterogeneous cancer of the lymphocytes (mostly of B-cells and mainly in people above 50) that comprises a range of pathological conditions characterized by a monoclonal expansion of phenotypically mature, but immunologically immature B-cells in the blood (Elphee, 2007).
  • diagnosis is based on three main parameters: (i) the mutational status of the B-cell receptor (BCR), (ii) the expression of the T-cell receptor signaling molecule ZAP70 and (iii) the presence of CD38 on the B-cell surface.
  • Prognostic markers include thymidine kinase, soluble CD23 and 2-microglobulin as well as chromosomal abnormalities on chromosome 6, 14, 13, 1 1 and 17 and sometimes trisomy 12.
  • CLL can stay untreated and is rarely the direct cause of death in the patient.
  • the VTIAQGGVLPNIQAV peptide generated in vivo has an m/z value of 740.4 Da.
  • the intensity in the top figure A is more than twice as high compared to the bottom figure B, and that ii) although there appears less labeled peptide present in the top figure, both patient samples are spiked with the same amount of labeled peptide, it is clear that much more peptide has been generated in the top figure A compared to the bottom figure B. In other words, significantly more H2A has been cleaved in the patient with severe CLL compared to the patient with less severe CLL.
  • the present invention provides a superior, quicker and cheaper diagnostic tool based on a clear-cut marker which correlates with disease severity and prognosis.
  • the present invention describes both the presence and amount of enzymatic cleavage of the carboxy-terminal tail of the histone H2A in between Valinen 4 and Leucine ⁇ in CLL B-cells as well as the use of a specific peptide having the amino acid sequence VTIAQGGVLPNIQAV that arises from said specific cleavage as a diagnostic and prognostic tool in clinical analysis of CLL.
  • VTIAQGGVLPNIQAV The absolute quantity of the peptide "VTIAQGGVLPNIQAV" in a tryptic digest of the genome from B-cells taken from CLL patients -which comprises only K- and R-terminal peptides- is a measure for the amount of genes in the genome that are potentially affected (activated).
  • VTIAQGGVLPNIQAV can, for example, be obtained via digesting said histone H2A ID which has firstly been cleaved in vivo in between Valine ⁇ and Leucine ⁇ as described above- with trypsin.
  • 'peptide' is meant a short polymer formed from the linking, in a defined order, of a- amino acids.
  • Peptide chains are usually short enough ( ⁇ 100 amino acids) to be made synthetically from the constituent amino acids.
  • the peptide of the present invention can also be obtained using any synthetic and/or recombinant method known in the art.
  • amino acids or amino acid residues are abbreviated as follows: alanine (Ala, A), asparagine (Asn, N), aspartic acid (Asp, D), arginine (Arg, R), cysteine (Cys;C), glutamic acid (Glu, E), glutamine (Gin, Q), glycine (Gly, G), histidine (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
  • Variants of the peptide "VTIAQGGVLPNIQAV" having similar biochemical or biophysical properties -especially peptides having an identical mass and retention time during liquid chromatography- are also encompassed by the present invention.
  • peptides consisting of the same 15 amino acids as the peptide VTIAQGGVLPNIQAV but wherein some of all of said amino acids have a different position within said peptide when compared to the position they have within the peptide VTIAQGGVLPNIQAV are also encompassed by the present invention.
  • the present invention further relates to a peptide consisting of the amino acid sequence VTIAQGGVLPNIQAV or a variant sequence thereof wherein said peptide is labeled, preferably isotopically labeled.
  • the amino acid proline of said peptide can comprise 5 isotopes C 13 and 1 isotope N 15 .
  • the latter labeling results in a mass difference of 6Da compared to the unlabeled peptide, which on a mass spectrum of an electrospray ionized sample results, in a difference of 3 m/z units.
  • Any suitable method may be used for labeling or differential isotope labeling of peptides as, for example, described in Ong SE, Foster LJ, Mann M. 2003: Methods: 124-30. Label-specific peptides allow one to quantify the differences in relative abundance between samples.
  • ESI nanospray ionization
  • iTRAQTM a commercially available stable isotope labeling system
  • the iTRAQTM labeling system allows selective labeling of up to eight different samples which are distinguished from one another in the mixture by MS/MS analysis.
  • the use of an isotopically labelled synthetic peptide and optimized LC/MS parameters also allows for absolute MS-quantification using the AQUATM-approach (Sigma Aldrich, St Louis, MO, USA) (Gerber et.al. 2003, PNAS).
  • the term 'mass spectrometer' refers to a device able to volatilize/ionize analytes to form gas- phase ions and to determine their absolute or relative molecular masses. Suitable forms of volatilization/ionization are electrospray, laser/light, thermal, electrical, atomized/sprayed and the like, or combinations thereof. Suitable forms of mass spectrometry include, but are not limited to, ion trap instruments, quadrupole instruments, electrostatic and magnetic sector instruments, time of flight (TOF) instruments, Fourier-transform mass spectrometers, and hybrid instruments composed of various combinations of these types of mass analyzers. These instruments may, in turn, be interfaced with a variety of sources that fractionate samples (for example, liquid chromatography or solid-phase adsorption techniques based on chemical or biological properties).
  • TOF time of flight
  • the present invention thus relates to the use of the above indicated peptide as a biomarker to evaluate the prognosis of a patient with CLL in vitro.
  • a biomarker' is meant a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.
  • said biomarker can be used, among other uses, to: 1) diagnose CLL; 2) evaluate the prognosis of CLL which encompasses predictions about the likely course of disease or disease progression, particularly with respect to the likelihood of metastasis, disease remission, disease relapse, tumor recurrence and death; 3) therapeutically stratify patients with CLL (i.e.
  • the present invention relates to the latter usages, wherein an increased presence (i.e. about a doubling of its presence) of the peptide of the present invention in a patient sample compared to a control sample of a healthy individual indicates the presence of CLL, and, wherein a dramatically increased level of the peptide of the present invention in a patient sample (i.e. of about 8 times), compared to a control sample, indicates CLL with a poor prognosis.
  • a patient sample' includes, but is not limited to, a primary tumor sample, circulating cancer cells or a biofluid such as blood, serum, plasma lymph, saliva or any other bodily secretion or derivative thereof. Methods for collecting various samples are well known in the art.
  • the terms 'an increased level in a patient sample, compared to a control sample' depends on how this level is measured.
  • the presence and/or amount of the biomarker in a biological sample of the present invention may be determined using any suitable assay capable of detecting the amount of the biomarker.
  • assay methods include, but are not limited to, mass spectrometry as indicated above, liquid chromatography, thin layer chromatography, fluorometry, radioisotope detection, affinity detection, and antibody-based detection such as Western Blot or immunohistochemical methods.
  • mass spectrometry biological samples may be obtained and used directly or may be separated in subfractions.
  • the CLL-associated proteins are digested into peptides with any suitable enzyme such as trypsin, which cleaves adjacent to lysine or arginine residues in proteins.
  • the peptides are then analyzed by a mass spectrometry method such as MALDI-TOF-MS or M/MS (solid phase), liquid chromatography (LC)-MS or MS/MS, and AQUATM, iTRAQTM, ICAT, or other forms of isotope tagging.
  • a 'control sample' is meant a similar sample as indicated above taken from a healthy patient not having CLL. Said control sample is important to determine the range of concentrations of the peptide VTIAQGGVLPNIQAV present in samples of healthy individuals.
  • the present invention relates to using the peptide of the present invention to determine cleavage of histone H2A, such as H2A ID, in cells.
  • H2A histone H2A
  • the present invention relates to using the peptide of the present invention to determine cleavage of histone H2A, such as H2A ID, in cells.
  • H2A Lys i i9-ubiquitination regulatory mechanisms that control double stranded DNA break repair, cell cycle regulation, apoptotic response, transcriptional activity of (hox-) genes and general cell differentiation (9-12)- the present invention might be used to detect the latter impairments.
  • the present invention relates to using the peptide of the present invention as a marker to diagnose or to determine the prognosis of chronic lymphocytic leukemia.
  • the presence and/or amount of said peptides in B cells of CLL patients or patients suspected to have CLL is determined by mass spectrometry, and more preferably wherein the mass spectrometry- quantification uses the AQUATM approach.
  • the present invention further relates to the usage of any proteolytic product of histone H2A or variants of histone H2A, or a fragment or variant of said proteolytic product, as a marker to diagnose or determine the prognosis of chronic lymphocytic leukemia, wherein said histone H2A has been cleaved in between Valine] j 4 and Leucine] 15 and wherein said variant of histone H2A has been cleaved in a position corresponding to Valine ⁇ and Leucine ⁇ when aligning histone H2A with said variant of histone 2A.
  • the latter fragments or variants of the proteolyic products must be capable, as for the above-described peptides of the present invention, to be used as a marker for CLL; i.e. a significantly higher amount of marker is found in CLL cases where the BCR is unmutated and expression of ZAP70 and CD38 is high in comparison to CLL cases where the BCR is mutated BCR and the expression of ZAP70 and CD38 is low without chromosomal aberrations and/or in comparison to control cases.
  • the peptide is about twice as abundant in mutated versus healthy B-cells and 4 times more abundant in unmutated vs mutated CLL.
  • BCR- and TCR-recombination and hypermutation in the DNA could be the process during which the cleavage of histone H2A in between Valinen 4 and Leucine] 15 is evolutionary relevant by avoiding double stranded DNA break repair, implying that the marker of the present invention is also involved in a broader spectrum of T- and B-cell cancers.
  • the present invention further relates to the usage of peptides according to the present invention claims to determine cleavage of histone H2A or variants of H2A in cancer cells, in particular in T and B- cancer cells.
  • the present invention also relates to a diagnostic kit comprising a peptide according to the present invention.
  • a diagnostic kit comprising a peptide according to the present invention.
  • Such a “CLL diagnostic kit” comprises for example the AQUA-reference labeled peptide, LC buffers and a user's manual with LC/MS parameters and cut-off values of the peptide concentration with predictive implications.
  • the term 'kit' refers to any manufacture (e.g. a package or a container) comprising at least one reagent (e.g. a labeled peptide, a buffer, etc.) for performing an assay which specifically detects the presence and amount of any proteolytic product of histone H2A or variants of histone H2A such as the specific peptides of the present invention.
  • kits can be included in the kits to validate the activity and correct usage of reagents employed in accordance with the present invention.
  • the design and use of controls is standard and well within the routine capabilities of those of ordinary skill in the art.
  • the kit can be promoted, distributed, or sold as a unit for performing the methods or usages of the present invention. Additionally, the kits can contain a package insert describing the kit and methods/usages for its use.
  • In-solution digest resuspend sample in 20 ⁇ 1 0.5M Triethylammonium bicarbonate (TEABC) buffer, (can be made by gassing C0 2 through a 5M TEA solution to a pH of 7.5-8.5); add ⁇ ⁇ Denaturant (2% SDS); add 2 ⁇ 1 Reducing reagent (50 mM TCEP or l OmM DTT) and incubate for lh @ 60°C; add ⁇ of a 200mM MMTS in isopropanol; incubate for lOmin @ RT; and, reconstitute a complete trypsin glass vial in 40 ⁇ 1 TEABC buffer and add ⁇ to each sample. Incubate o/n @ 37°C.
  • TEABC Triethylammonium bicarbonate
  • iTRAQ labelling reconstitute each label vial in 70 ⁇ EtOH; add label to samples (final organic content should be >65%); vortex and spin and incubate for lh @ RT; add 100 ⁇ of MQ H 2 0 to hydrolyze unreacted label. Alternatively add 6 ⁇ 1 of a 5% Hydroxylamine solution; and, combine samples and dry in speed vac c. Purify sample
  • Step I SCX cartridge, (check cartridge retention capacity by first "purifying" lpmol of Pep Mix! Analyse on MS: a minimum of 40 peptides should be identified on the 63min gradient on the HPLC, 7 channels in MS/MS)
  • Buffer C 5 mM KH2P04; 500 mM KCl; 25% ACN; pH 2.7
  • the peptides will have better retention and fraction 1 1 and 12 should be kept separated (or might be overloaded when combined). Gradient might be optimized based on first run; different sample content could demand different fractionation conditions, especially non-cellular-derived samples
  • histones are firstly purified as follows:
  • PBS can be supplemented with 5mM sodium butyrate to retain levels of histone acetylation
  • TEB Triton Extraction Buffer
  • PMSF phenylmethylsulfonyl fluoride
  • NaN3 Triton Extraction Buffer

Abstract

La présente invention concerne des produits protéolytiques d'histone H2A. En particulier, la présente invention concerne un peptide ayant la séquence d'acides aminés VTIAQGGVLPNIQAV qui peut être utilisé pour déterminer le clivage d'histone H2A. Plus particulièrement, la présente invention concerne ce dernier peptide en tant que marqueur pour diagnostiquer ou déterminer le pronostic d'un cancer tel que la leucémie lymphoïde chronique.
PCT/EP2010/063425 2009-09-14 2010-09-14 Produit protéolytique d'histone h2a en tant que marqueur pour le cancer WO2011029946A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103529138A (zh) * 2013-07-23 2014-01-22 浙江省疾病预防控制中心 一种牛β-酪蛋白定量检测试剂盒及其应用
US11391742B2 (en) 2015-02-10 2022-07-19 B.R.A.H.M.S. Gmbh Free histone proteins as biomarkers

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103529138A (zh) * 2013-07-23 2014-01-22 浙江省疾病预防控制中心 一种牛β-酪蛋白定量检测试剂盒及其应用
US11391742B2 (en) 2015-02-10 2022-07-19 B.R.A.H.M.S. Gmbh Free histone proteins as biomarkers

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