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WO2020239947A1 - Procédé de pronostic de la leucémie - Google Patents

Procédé de pronostic de la leucémie Download PDF

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WO2020239947A1
WO2020239947A1 PCT/EP2020/064909 EP2020064909W WO2020239947A1 WO 2020239947 A1 WO2020239947 A1 WO 2020239947A1 EP 2020064909 W EP2020064909 W EP 2020064909W WO 2020239947 A1 WO2020239947 A1 WO 2020239947A1
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proteins
seq
leukemic
sumoylation
ubiquitination
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Guillaume BOSSIS
Pierre GATEL
Marc Piechaczyk
Christelle REYNES
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Montpellier
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Montpellier
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    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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 invention relates to a prognosis of leukemia, in particular a prognosis of resistance to drug that can be used to treat leukemic patients.
  • the inventors identified a modifomic signature based on protein post-translational modifications by Ubiquitin and the Ubiquitin-like SUMO polypeptides that may allow rapid discrimination of chemosensitive versus chemoresistant Acute Myeloid Leukemia (AML) at diagnosis.
  • AML Acute Myeloid Leukemia
  • One aim of the invention is to provide a method that allows the practitioner to determine the best therapy to carry out faced to a patient.
  • Another aim of the invention is to provide kits that could help the practitioner’s choice when intending to provide the best therapy to the patients.
  • the invention relates to a method for, or of, determining, preferably in vitro , drug-resistance of cells of a leukemic sample obtained from a patient afflicted by a leukemia, said method comprising:
  • said at least 4 proteins correspond to the proteins consisting essentially or consisting of the sequences SEQ ID NO: 1 to 4, wherein said 23 proteins correspond to the proteins consisting essentially or consisting of the sequences SEQ ID NO: 1 to 23, and wherein said group of 122 proteins corresponds to the proteins consisting essentially or consisting of the sequences SEQ ID NO: 1 to 122,
  • said reference level corresponding to the mean of the level of modification by ubiquitination and/or sumoylation of each at the least 4 proteins of several, possibly independent, leukemic cells that are sensitive to said drug;
  • the inventors identified a group of 122 proteins that constitute group of proteins that are modified by ubiquitin/SUMO proteins and said modification being informative regarding the resistance to a drug. More particularly, the information which is important in the variation of the modification of said proteins by SUMO or ubiquitin.
  • the group of 122 proteins according to the invention consists of the following proteins:
  • DCUN1D1 BC009478.1 IOH12273 DCUN1D1 SEQ ID NO: 8 chromosome 9 open reading frame 71 (C9orf71) NM_153237.1 IOH22672 C9orf71 SEQ ID NO: 9 polymerase (RNA) III (DNA directed) polypeptide K, 12.3 kDa (POLR3K) NM_016310.2 IOH12571 POLR3K SEQ ID NO: 10 ring finger protein 34 (RNF34), transcript variant 2 NM_025126.2 IOH6675 RNF34 SEQ ID NO: 11 zinc finger, MYM-type 5 (ZMYM5) BC007048.1 IOH7230 ZMYM5 SEQ ID NO: 12 arrestin domain containing 1 (ARRDC1) NM_152285.1 IOH21698 ARRDC1 SEQ ID NO: 13 hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) NM_004712.3 IOH4952 HGS SEQ ID NO: 9
  • RAD23A NM_005053.2 IOH22983 RAD23A SEQ ID NO: 80 Leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 4 (LILRA4), mRNA NM_012276.3 IOH43377 LILRA4 SEQ ID NO: 81 Potassium voltage-gated channel, shaker-related subfamily, member 4 (KCNA4), mRNA NM_002233.1 IOH29649 KCNA4 SEQ ID NO: 82 rhomboid domain containing 1 (RHBDD1) BC027900.1 IOH27306 RHBDD1 SEQ ID NO: 83 RAD18 homolog (S.
  • the first step intends to evaluate the modification of at least the proteins consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, by ubiquitin or SUMO, or ubiquitin and SUMO proteins, in order to obtain a value of modification by one or two of said Ubiquitin and SUMO proteins.
  • Such modifications by ubiquitin or SUMO can be easily evaluated by immunoblotting assays using specific antibodies directed respectively against Ubiquitin or SUMO proteins. These proteins are the markers of the activity of the enzymes involved in the Ubiquitin/Sumo pathways, which are present in the leukemic samples.
  • the at the least 4 proteins chosen among the group of 23 proteins, said group of 23 proteins belonging to a set of 122 proteins, are contacted with the leukemic samples such that the deregulation of the Ubiquitin/Sumo pathways in the leukemic sample is detected by variations in the levels of modification by ubiquitination or sumoylation of said at least 23 proteins.
  • the proteins which are modified by ubiquitin and/or SUMO proteins will therefore interact with antibodies directed against ubiquitin and/or SUMO proteins to form a molecular complex.
  • the complexes can be detected by secondary antibodies that recognize (and interact with) constant part FC chain of said antibodies directed against ubiquitin and/or SUMO proteins.
  • Said complex can be identified when the secondary antibodies are labeled with reporter molecule, such as fluorescent protein, peroxidase, fluorescent dyes etc...
  • reporter molecule such as fluorescent protein, peroxidase, fluorescent dyes etc...
  • Extracts are finally centrifuged twice (16000 g) at 4°C for 20 min and supernatants were aliquoted, flash-frozen and kept at -80 °C until use.
  • SUMO-1-, SUMO-2- and Ubiquitin vinyl sulfones (0.5 ⁇ M each) are added to 10 ⁇ L of cellular extracts and incubated at 4 °C for 15 min.
  • Beads are washed twice for 5 min with PBS containing 0.05 % Tween-20 and 0.5 % SDS and 3 times for 5 min with PBS containing 0.05 % Tween-20. They are then incubated with 1 ⁇ g/mL of anti-SUMO-1 (21C7) and anti-Flag antibodies for 1 hr under agitation at room temperature. After washing in PBS containing 0.05% Tween-20 for 5 min, they are incubated for 30 min at room temperature with anti-mouse Alexa Fluor 488- and anti-rabbit Alexa Fluor 405 antibodies in 100 ⁇ L of PBS containing 0.05 % Tween-20. Beads are again washed for 5 min with PBS containing 0.05 % Tween-20. They are then resuspended in 200 ⁇ L PBS and flow-cytometry-analysed.
  • the detection of the above-mentioned complexes allows to establish a value of modification which corresponds to the Ubiquitin/SUMO modification status of said at least 4 proteins for each leukemic samples of unknown response to said drug.
  • step a In order to normalize the value calculated in the step a), it is also calculated the value of the modification by ubiquitin and SUMO of said at least 4 proteins in leukemic cells that are known to be sensitive to said drug. These cells correspond to the reference cells.
  • Drug sensitivity of said control cells can be determined experimentally, or is known either by nature (the cells were selected to be sensitive to said drug), or can be obtained from patient afflicted by a leukemia that was successfully treated with said drug.
  • Said value can be established for each protein as VRi, wherein i corresponds to the SEQ ID of said at least 4 proteins.
  • Ri VCi/VRi, wherein i corresponds to the SEQ ID of said at least 4 proteins.
  • the ratio R1, R2, R3 and R4 are calculated. If R1, R2, R3 and R4 are between 0.8 and 1,25, then the leukemic cells are considered to be sensitive to said drug.
  • the ratio can represent an increase of the ubiquitin/SUMO modification in the leukemic cells resistant to said drug compared to the control leukemic cells that are sensitive to said drug.
  • an increase of the modification by ubiquitin or SUMO can represent the hallmark of resistance.
  • the ratio can also represent a decrease of the ubiquitin/SUMO modification in the leukemic cells resistant to said drug compared to the control leukemic cells that are sensitive to said drug.
  • a decrease of the modification by ubiquitin or SUMO can represent the hallmark of resistance.
  • the invention relates to the method as defined above, wherein
  • the inventors identified that the protein as set forth in SEQ ID NO: 1 is only modified by SUMO protein. Therefore, the modification that can be evaluated for this protein corresponds only to an increase or a decrease of the amount of SUMO protein.
  • the inventors also identified that the protein as set forth in SEQ ID NO: 2 is only modified by ubiquitin protein. Therefore, the modification that can be evaluated for this protein corresponds only to an increase or a decrease of the amount of ubiquitin protein.
  • the invention relates to the method as previously defined, wherein if the modification of the protein as set forth in SEQ ID NO: 2 or 3 or 4 is an ubiquitination, and if the ratio is higher than 1.25, then leukemic sample will be resistant to said drug.
  • the proteins as depicted in SEQ ID NO: 2, 3 and 4 are modified by ubiquitin. Therefore, the evaluation of the variation of ubiquitination of at least one of these proteins gives information regarding the resistance to said drug.
  • the invention relates to the method as defined above, wherein if the modification of the protein as set forth in SEQ ID NO: 1 or 3 or 4 is a sumoylation, and if the ratio is higher than 1.25, then leukemic sample will be resistant to said drug.
  • the invention relates to the method as defined above, comprising a step a) of evaluating, in said leukemic sample, the level of modification by ubiquitination and/or sumoylation of each protein of said group of 23 proteins, said group of 23 proteins belonging to a set of 122 proteins.
  • the inventors identified that if a group of 23 proteins is studied, i.e. if the variation of the amount of ubiquitin and/or SUMO of at least said 23 proteins represented by the proteins as set forth in SEQ ID NO: 1 to 23, the evaluation of the drug resistance or sensitivity is higher.
  • an advantageous embodiment of the invention relates to a method for, or of, determining, preferably in vitro , drug-resistance of cells of a leukemic sample obtained from a patient afflicted by a leukemia, said method comprising:
  • said at least 23 proteins correspond to the proteins consisting essentially or consisting of the sequences SEQ ID NO: 1 to 23, and wherein said group of 122 proteins corresponds to the proteins consisting essentially or consisting of the sequences SEQ ID NO: 1 to 122,
  • said reference level corresponding to the mean of the level of modification by ubiquitination and/or sumoylation of each at the least 23 proteins of several, possibly independent, leukemic cells that are sensitive to said drug;
  • SEQ ID NO: 2 the proteins consisting essentially or consisting of the respective sequences as set forth in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 17 to 19, SEQ ID NO: 21 and SEQ ID NO: 23 are modified by Ubiquitination, and
  • SEQ ID NO: 3 and 4 the proteins consisting essentially or consisting of the respective sequences as set forth in SEQ ID NO: 3 and 4, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 22 are modified by Ubiquitination and Sumoylation.
  • the invention relates to the method as defined above, comprising a step a) of evaluating, in said leukemic sample, the level of modification by ubiquitination and/or sumoylation of each protein of said set of 122 proteins.
  • the inventors identified that the evaluation of ubiquitination/SUMOylation of the 122 proteins as depicted in SEQ ID NO: 1 to 122 gives the best results in determining said drug resistance.
  • the invention relates advantageously to a method for, or of, determining, preferably in vitro , drug-resistance of cells of a leukemic sample obtained from a patient afflicted by a leukemia, said method comprising:
  • said reference level corresponding to the mean of the level of modification by ubiquitination and/or sumoylation of each 122 proteins of several, possibly independent, leukemic cells that are sensitive to said drug;
  • the invention relates to the method as defined above, wherein said drug is chosen from anthracycline and/or cytarabine.
  • the invention relates to the method as defined above, wherein said leukemia is an acute myeloid leukemia.
  • the invention relates to a method for, or of, the prognosis, preferably in vitro, of a leukemia in a sample of a patient comprising the following steps:
  • said at least 4 proteins corresponds to the proteins consisting essentially or consisting of the sequences SEQ ID NO: 1 to 4
  • said 23 proteins corresponds to the proteins consisting essentially or consisting of the sequences SEQ ID NO: 1 to 23
  • said group of 122 proteins corresponds to the proteins consisting essentially or consisting of the sequences SEQ ID NO: 1 to 122
  • the leukemia is an acute myeloid leukemia.
  • the invention also relates to a kit comprising:
  • the invention relates to the kit as defined above, wherein said determined drug is anthracyclin and/or cytarabine.
  • the invention relates advantageously to a kit comprising:
  • UbL biomarkers of AML chemoresistance The level of modification of the proteins modified by Ubiquitin (left panel) or SUMO-1 (central panel) selected in C was compared between the parental HL-60 and U937 and their resistant counterparts.
  • Right panel Venn diagram with proteins showing a significant p-value in both Wilcoxon signed-rank test and one sample t -test and a ratio between resistant and parental cells higher than 1.25 or lower than 0.8.
  • Figure 11 represents a graph showing the probability of acute myeloid leukemias sensitivity/resistance using a score based on the use of a genetic algorithm and a linear discriminant analysis. This score was used to predict the probability of resistance for the U937 and HL60 cell lines. Cells were considered sensitive to chemotherapy if the probability to belong to the group of resistant cells was below 50% and resistant if over 50%.
  • Figure 14 represents the quantification of the modifications.
  • background cell extracts supplemented with NEM to inhibit UbL conjugation activities
  • the inventors first developed cellular models of resistance to Ara-C or DNR using two reference chemosensitive AML cell lines, HL-60 and U937. Generated Ara-C- (Ara-C-R) and DNR-resistant (DNR-R) sublines showed significantly higher IC 50 ( Figure 1 ). To identify UbL substrate biomarkers of AML chemoresistance, the inventors then resorted to the Protoarrays from Life Technologies, which display more than 9000 recombinant human proteins spotted in duplicate on a nitrocellulose-coated slide. Such arrays have already been used successfully to identify substrates of certain E3 Ubiquitin ligases using either total cell extracts or recombinant enzymes.
  • the inventors then identified the proteins, which were differentially modified by the cell extracts from the resistant cell lines compared to the parental ones.
  • a first analysis was performed by comparing the data for all Ara-C-R or DNR-R cells with those from parental HL60 and U937 cells. This led to the identification of 52 proteins differentially modified by Ubiquitin and 27 proteins differentially modified by SUMO-1 ( Figure 4 ).
  • the inventors then performed a second analysis in which the data for each cell line and each resistance (DNR and Ara-C) were considered separately.
  • the inventors selected 23 of the 122 proteins that showed both a high level of modification and the most robust signal differences between sensitive and resistant cell lines ( proteins of sequences SEQ ID NO: 1 to SEQ ID NO: 23 ).
  • the inventors used them to generate a UbL score aiming at predicting patients' response to chemotherapy. Using this score, they could predict whether AML cell line were sensitive or resistant to DNR or Ara-C in 16 cases out of 18 tested (6 cell lines, 3 replicates) ( Figure 11 ).
  • they resorted to bone marrow aspirates from 4 patients, all of them being of the rather immature M1 subtype in the FAB classification.
  • the inventors cloned 15 proteins of the signature (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 ) in frame with GST, produced them in bacteria and coupled them to differently colored XMap beads.
  • the coupled beads were then multiplexed (up to 10 different beads per tube) and incubated with extracts from parental-, Ara-C-R- or DNR-R HL60 cells before flow cytometry analysis using anti- SUMO-1 or Flag tag to quantify modification by SUMO and Ubiquitin, respectively ( Figure 7 ). If ubiquitylation was detected on all of the proteins tested, this was unfortunately not the case for SUMOylation. A most likely explanation is that Ubiquitin can make long polyUbiquitin chains, facilitating its detection on protein substrates, whereas SUMO cannot.
  • the inventors work identifies a new class of modifomic biomarkers that might be used at diagnosis, in combination with other biomarkers and clinical data, to help clinicians predict AML patient response to anthracyclines and cytarabine.
  • the assay the inventors have miniaturized was shown adapted to patient sample analysis, paving the way to novel clinical developments.
  • HL-60 and U937 cells were cultured at 37 °C in RPMI medium supplemented with 10 % fetal bovine serum (FBS) and streptomycin/penicillin in the presence of 5 % CO 2 . Both cell lines were authenticated by the ATCC using Short-Tandem-Repeat analysis. All cells were regularly tested negative for mycoplasma. After thawing, cells were passaged at a density of 3.10 5 /ml every 2-3 days for no more than 10 passages. U937 and HL-60 cells resistant to Ara-C (cytarabine) and DNR were generated by culture in the presence of increasing drug concentrations (up to 0.1 ⁇ M for Ara-C and 0.03 ⁇ M for DNR) for 2-3 months.
  • FBS fetal bovine serum
  • Cells were seeded at a concentration of 2.10 5 /mL in RPMI medium complemented with 0.1, 1, 10, 50, 100 or 250 ⁇ M of Ara-C (Sigma-Aldrich) or 0.01, 0.05, 0.1, 0.5, 1 or 10 ⁇ M of daunorubicin (Sigma-Aldrich). Viability was measured 24 hrs later using the MTS assay from Promega following the manufacturer’s protocol. IC 50 were calculated using the GraphPad PRISM software.
  • Cells grown at a 5-8.10 5 /mL density were spun down (300 g) at 4 °C for 5 min and washed once with PBS. After pellet resuspension in 1 mL of PBS, they were centrifuged again (16,000 g) at 4 °C for 5 min.
  • Pellets were resuspended and incubated at 4 °C for 30 min in a hypotonic buffer (20 mM HEPES pH 7.5, 1.5 mM MgCl 2 , 5 mM KCl, 1 mM DTT and 1 mg/L of aprotinine, leupeptin and pepstatin) in a volume of either (i) 100 ⁇ L per 50.10 6 cells to generate concentrated extracts used for ProtoArray probing or (ii) 25 ⁇ L per 2.10 6 cells in the case of extracts used in XMap bead-based flow cytometry assays.
  • a hypotonic buffer (20 mM HEPES pH 7.5, 1.5 mM MgCl 2 , 5 mM KCl, 1 mM DTT and 1 mg/L of aprotinine, leupeptin and pepstatin
  • Cell lysis was achieved through 4 freezing/thawing cycles using liquid nitrogen and DNA was sheared owing to 10 passages through a 20-1/2G needle. Extracts were finally centrifuged twice (16000 g) at 4°C for 20 min and supernatants were aliquoted, flash-frozen and kept at -80 °C until use.
  • the extracts were then supplemented with home-made recombinant SUMO-1 and SUMO-2 (15 ⁇ M, produced as previously described), 30 ⁇ M Flag-ubiquitin (Boston Biochem), 15 ⁇ M NEDD8 (Boston Biochem), 0.1% Tween-20, and 1 mM ATP Immediately after, they were laid on ProtoArray slides, which were covered with a coverslip and incubated at 30 °C for 1 hr in a humidified atmosphere. Arrays were washed 3 times for 5 min with a washing buffer (PBS pH 7.4, 0.1 % Tween 20, 1X Synthetic Block) supplemented with 0.5 % SDS and, then, twice for 5 min with only the washing buffer.
  • a washing buffer PBS pH 7.4, 0.1 % Tween 20, 1X Synthetic Block
  • Arrays were washed 5 times for 5 min with the washing buffer, once with H 2 O and, finally, dried by centrifugation before fluorescence scanning using the Innoscan 710 device from Innopsys. The arrays were then stripped with 200 mM Glycine, 0.5% SDS, pH 2.2 for 20 min and probed again with anti-SUMO-2 (8A2 from the Developmental Studies Hybridoma Bank) and anti-Nedd8 (Y297, Abcam) antibodies. However, some signals were remaining from the first hybridization. To avoid false positive, the inventors thus decided to consider only the signals obtained for SUMO-1 and Ubiquitin.
  • Measured fluorescence intensities were associated to the corresponding protein ID according to their coordinates on the arrays using the Mapix software (Innopsys). Intensities were processed using the PAA R package. In brief, (i) duplicated protein spot intensities were averaged using the Load GPR function and (ii) the background was corrected using the Background Correct function. Fluorescence intensities within the same experiment were normalized by quantiles using the Normalize Array function. Finally, intensities were normalized between the different experiments using the Batch Adjust function.
  • the inventors To identify the proteins modified by Ubiquitin or SUMO-1 on the ProtoArrays, the inventors first had to filter proteins proteins displaying signal significantly higher in UbL conjugation-permissive- than those in UbL conjugation non-permissive conjugation conditions (i.e. control conditions using NEM-treated extracts). With regard to the statistical test adapted to the exploitation of the inventors’ results, the classical Student t-test was not recommended, as variances could be very different between UbL conjugation permissive and non-permissive conditions. The inventors therefore selected proteins with significant p-values (lower than 0.05) in both the parametrical Welch- and the non-parametrical Wilcoxon-Mann-Whitney (WMW) test. Then, proteins having an averaged normalized fluorescence intensity value less than 800 were filtered out in order to obtain a list of robustly modified proteins.
  • WMW non-parametrical Wilcoxon-Mann-Whitney
  • the inventors decided to work on ratios between the parental and drug-resistant cell samples for each protein.
  • the inventors thus compared, for each of the modified proteins (NEM-filtering), the ratios between sensitive and resistant cells using both a Wilcoxon signed-rank test and a one sample t -test.
  • This analysis was first performed on all arrays (both cell lines and drugs combined). In this case, the sample size being large, the inventors considered as differentially modified all proteins showing p-values ⁇ 0.05 in both tests.
  • the sample size being smaller, the inventors considered as differentially modified the proteins showing p-values ⁇ 0.05 in the one sample t-test.
  • cDNA encoding for the proteins of interest were recovered from the Ultimate ORF library (Thermofisher) and cloned in the bacterial expression vector pGGWA vector using the Gateway technology according to manufacturer’s protocol (Life Technologies). Constructs were then transformed in BL21 (DE3) E. coli strain. Protein production was induced with 1 mM isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG) for 6 hrs in exponentially growing bacteria at 25°C.
  • IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • Bacterial pellets were resuspended in 50 mM Tris-HCl pH 8.6 containing 500 mM NaCl and 50 mM MgSO 4 , and flash-frozen in liquid N 2 . After thawing, bacterial suspensions were supplemented with 1 mg/mL lysozyme (Sigma-Aldrich), 8 mM ⁇ -mercaptoethanol, 1 mg/L aprotinin, leupeptin and pepstatin and incubated at 4°C for 1 hr. Bacterial debris were spun down (100 000 g for 1 hr).
  • the extract was then bound to Glutathion agarose beads (Generon) equilibrated in Tris 50 mM pH 8.6, NaCl 500 mM, MgSO4 50 mM, 8 mM ⁇ -mercaptoethanol, 1 mg/L aprotinin, leupeptin, pepstatin.
  • the column was then extensively washed with Tris 50 mM pH 8.6, NaCl 150 mM, MgSO4 50 mM, 8 mM ⁇ -mercaptoethanol, 1 mg/L aprotinin, leupeptin, pepstatin and eluted by addition of 20 mM reduced glutathione (Sigma-Aldrich).
  • Beads were then washed in PBS containing 500 mM NaCl and incubated with 7 ⁇ g of recombinant protein to be coupled in 100 ⁇ L PBS at room temperature for 2 hrs. They were then washed twice with PBS containing 0.1 % BSA, 0.02 % Tween 20, 0.05 % sodium azide and 500 mM NaCland stored at 4 °C in PBS containing 0.1 % BSA, 0.02 % Tween-20, 0.05 % sodium azide.
  • SUMO-1-, SUMO-2- and Ubiquitin vinyl sulfones (0.5 ⁇ M each) were added to diluted cellular extracts (10 ⁇ L), which were incubated at 4 °C for 15 min. Control extracts were also incubated with 50 mM NEM.
  • the inventors then added to the extract 10 3 protein-coupled XMap beads contained in 10 ⁇ L of a reaction buffer containing 20 mM HEPES pH 7.3, 110 mM KOAc, 2 mM Mg(OAc) 2 , 0.05 % Tween-20, 0.5 mM EGTA, 0.2 mg/mL ovalbumine, 1 mM DTT, 1 mg/L aprotinin, leupeptin and pepstatin, 1 mM ATP, 30 ⁇ M Flag-ubiquitin, 15 ⁇ M SUMO-1 and 15 ⁇ M SUMO-2. Reaction were performed at 30 °C for 45 min.
  • Beads were washed twice for 5 min with PBS containing 0.05 % Tween-20 and 0.5 % SDS and 3 times for 5 min with PBS containing 0.05 % Tween-20. They were then incubated with 1 ⁇ g/mL of anti-SUMO-1 (21C7) and anti-Flag antibodies for 1 hr under agitation at room temperature. After washing in PBS containing 0.05% Tween-20 for 5 min, they were incubated for 30 min at room temperature with anti-mouse Alexa Fluor 488- and anti-rabbit Alexa Fluor 405 antibodies in 100 ⁇ L of PBS containing 0.05 % Tween-20.
  • Age* Age at diagnosis
  • Blasts* Blasts at diagnosis in bone marrow
  • Blasts** Blasts 30 days after diagnosis in bone marrow

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Abstract

L'invention concerne un procédé de détermination in vitro de la résistance aux médicaments de cellules d'un échantillon leucémique, ledit procédé comprenant, une étape d'évaluation, dans des cellules dudit échantillon leucémique , du niveau de modification par ubiquitination ou sumoylation de chaque protéine d'au moins 4 protéines choisies parmi un groupe de 23 protéines, ledit groupe de 23 protéines appartenant à un ensemble de 122 protéines.
PCT/EP2020/064909 2019-05-29 2020-05-28 Procédé de pronostic de la leucémie Ceased WO2020239947A1 (fr)

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WO2022129180A1 (fr) * 2020-12-15 2022-06-23 Centre National De La Recherche Scientifique Sqstm1 et son utilisation dans le traitement du cancer
WO2024249881A3 (fr) * 2023-06-02 2025-05-01 BIOMILQ, Inc. Compositions d'ostéopontine et procédés d'utilisation

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