WO2020234482A1 - Signatures de résistance mapki - Google Patents

Signatures de résistance mapki Download PDF

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WO2020234482A1
WO2020234482A1 PCT/EP2020/064402 EP2020064402W WO2020234482A1 WO 2020234482 A1 WO2020234482 A1 WO 2020234482A1 EP 2020064402 W EP2020064402 W EP 2020064402W WO 2020234482 A1 WO2020234482 A1 WO 2020234482A1
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ptrf
melanoma
expression level
cells
threshold
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Verena PAULITSCHKE
Hubert Pehamberger
Rainer KUNSTFELD
Mitchell LEVESQUE
Ossia EICHHOFF
Christopher GERNER
Ruedi Aebersold
Reinhard DUMMER
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Universität Zürich
Medizinische Universität Wien
Universität Wien
ETH Zürich
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    • 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
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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 present invention relates to a method for attributing a probability to a cancer cell of being responsive to treatment with an inhibitor of a mitogen-activated protein kinase (MAPK) inhibitor.
  • the method relates to a method of assigning a patient to a treatment regime, and to the use of checkpoint inhibitor agents in the treatment of cancer patients predicated on the results of certain characteristic biomarker assays.
  • MAPK mitogen-activated protein kinase
  • BRAFi BRAF inhibitors
  • Proteomics has been used to predict pleiotropic acidophilic protein kinase CK2 (CK2) as a potential drug target, demonstrating that CK2 blockade potentiated the antiproliferative effects of BRAF and MEK inhibition in BRAF-mutated cancers (Parker R et al., 2014, Molecular cancer therapeutics, 13(7):1894-906).
  • Phosphoproteomics has shown changes in cytoskeletal regulation, GTP/GDP exchange, protein kinase C, insulin growth factor (IGF) signaling, and melanosome maturation after transition to a drug resistant phenotype (Parker R et al., 2015, Frontiers in oncology, 5:95).
  • BRAFi resistance is associated with a loss of differentiation, an enhanced expression of the lysosomal compartment, an increased potential for metastasis, and epithelial-mesenchymal transition (EMT) (Paulitschke V et al., 2015, Molecular cancer therapeutics, Epub 2015/01/24).
  • phenotype switching from a melanocytic to a mesenchymal state.
  • Invasive phenotype melanoma cells are resistant to BRAFi treatment and tend to downregulate lineage-specific genes (e.g. MelanA, MITF) while up-regulating factors known to be involved in drug resistance (e.g., Wnt5a).
  • TQRb chronic exposure of proliferative melanoma cells to TQRb causes a phenotype- switch which involves the activation of PI3K signalling, downregulation of E-cadherin, and the loss of tissue-specific marker gene expression, which is a process similar to EMT and contributes to melanoma heterogeneity.
  • the objective of the present invention is to provide means and methods to identify and discriminate responder and non-responder to MAPKi treatment and to establish new methods for the patient stratification. This objective is attained by the subject-matter of the claims of the present specification.
  • the problem underlying the present invention was to identify a proteomic signature of MAPKi resistance in melanoma cells, to mechanistically dissect the role of genes in that signature, and to validate the most informative features on patient biopsies prior to MAPKi-therapy.
  • high-throughput techniques such as sub-cellular shotgun proteome analyses in two different MS centres and RNAseq
  • the inventors identified two proteins associated with a mesenchymal phenotype and demonstrated the involvement of PTRF in the invasive phenotype associated with MAPKi resistance in melanoma. Further validation in patient biopsies suggests that PTRF expression is significantly highly expressed in patients who fail to respond to targeted therapy.
  • PTRF in the context of the present specification relates to polymerase I and transcript release factor, also known as Cavin-1 .
  • the Entrez Gene ID for PTRF is 2841 19.
  • IGFBP7 in the context of the present specification relates to insulin like growth factor binding protein 7.
  • the Entrez Gene ID for IGFBP7 is 3490.
  • ALDH1A1 and ALDH1A3 in the context of the present specification relate to isoforms 1 and 3, respectively, of the member of the family of aldehyde dehydrogenases. High expression of ALDH1A1 and ALDH1A3 is associated with resistance to MAPKi treatment.
  • kynurenin in the context of the present specification relates to the non-proteinogenic amino acid kynurenine, in particular to the biologically active stereoisomere (Sj-kynurenine, also known as L-kynurenine (CAS No. 2922-83-0).
  • tenascin-C in the context of the present specification relates to a member of the tenascin extracellular matrix glycoprotein family (TNC, Entrez Gene ID 3371 ). High expression of Tenascin-C is associated with resistance to MAPKi treatment.
  • MEK is synonymous with mitogen-activated kinase kinase (MPA2K, MAPKK) and in the context of the present specification relates to the isoforms 1 and 2, MEK1 and MEK2.
  • expression refers to the process by which DNA is transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and/or protein product.
  • the term when used in the context of cells, or cancer cells, or a cancer, the term refers to said cells significantly decreasing in growth rate, decreasing in vitality, and / or increasing with respect to markers of apoptosis or necrosis, when the patient suffering from a cancer characterized with such cells, cancer cells or cancer is treated with an effective dose of the compound.
  • the term When used in the context of a disease or the general welfare of a patient, the term relates to a significant extension of progress-free survival (PFS), overall survival (OS) or time to death (TTD) when the patient suffering from a cancer characterized with such cells, cancer cells or cancer is treated with an effective dose of the compound.
  • PFS progress-free survival
  • OS overall survival
  • TTD time to death
  • checkpoint inhibitory agent or checkpoint inhibitory antibody is meant to encompass an agent, particularly an antibody (or antibody-like molecule) capable of disrupting the signal cascade leading to T cell inhibition after T cell activation as part of what is known in the art the immune checkpoint mechanism.
  • a checkpoint inhibitory agent or checkpoint inhibitory antibody include antibodies to CTLA-4 (Uniprot P16410), PD-1 (Uniprot Q151 16), PD-L1 (Uniprot Q9NZQ7), B7H3 (CD276; Uniprot Q5ZPR3), Tim-3, Gal9, VISTA, Lag3.
  • checkpoint agonist agent or checkpoint agonist antibody is meant to encompass an agent, particularly but not limited to an antibody (or antibody-like molecule) capable of engaging the signal cascade leading to T cell activation as part of what is known in the art the immune checkpoint mechanism.
  • Non-limiting examples of receptors known to stimulate T cell activation include CD122 and CD137 (4- 1 BB; Uniprot Q0701 1 ).
  • the term checkpoint agonist agent or checkpoint agonist antibody encompasses agonist antibodies to CD137 (4-1 BB), CD134 (0X40), CD357 (GITR) CD278 (ICOS), CD27 and CD28.
  • a first aspect of the invention relates to a method for attributing a probability to a cancer cell sample. This probability quantifies the likelihood that the cells of the sample are responsive to treatment with a mitogen-activated protein kinase (MAPK) inhibitor.
  • the method comprises the steps of
  • this aspect of the invention can be formulated as a method of assigning to a cancer patient a likelihood of being responsive to, or to clinically benefit from, treatment with a MAPK inhibitor.
  • the method comprises the steps of
  • the method can be used inversely to identify patients who are unlikely to benefit from kinase inhibitor therapy, and who are therefore likely to profit more from immediate assignment to an alternative therapy, particularly checkpoint inhibitor therapy, of which at present antibodies to PD-1 and / or PD-L1 are currently the method of choice.
  • checkpoint inhibitor therapy of which at present antibodies to PD-1 and / or PD-L1 are currently the method of choice.
  • Other checkpoint inhibitor and checkpoint agonist agents are under development and are likely to be used in the clinic in a similar fashion.
  • Another alternative of this aspect of the invention relates to a method for predicting, or attributing a probability to, a cancer cell of being responsive to treatment with a MAPK inhibitor.
  • the method comprises the steps of
  • the threshold is determined for expression of PTRF by staining a histological sample with PTRF-specific antibodies and the threshold expression level is equal or smaller than 2.5, particularly ⁇ 2.2. In certain embodiments of this aspect, the threshold is determined for expression of IGFBP7 by determining the protein level in a serum sample and the threshold expression level is equal or smaller than 1 ng/ml. In certain embodiments, the expression is determined for both proteins and compared to the respective threshold.
  • Quantification of PTRF expression is performed by visual microscopic examination of stained tissue sections of a tumour biopsy. Staining is effected with a fluorescently labelled high-affinity antibody specific for PTRF. Each section is ranked according to standard pathological practice as belonging to one of four categories: 1 is assigned to samples showing ⁇ 50% staining, 2 is assigned to samples showing >50% up to 75%, 3 is assigned to samples showing > 75% and up to 100% and 4 was ranked for positive nuclear staining. Nuclear staining was found invariably associated to patients with low response to MAPK inhibitors.
  • the values obtained are used to compute a mean value for all tumour specimens examined. If only one section is examined, the cut-off value of 2 is applied instead of 2.2, and a cut-off value of 3 is applied instead of 3.3.
  • expression of a biomarker is determined by nucleic acid quantification, particularly RNA quantification.
  • Another aspect of the invention relates to a method for assigning a patient suffering from a cancer to treatment with an immune checkpoint inhibitor (or checkpoint agonist) agent, wherein the method comprises the steps of
  • the threshold is determined for expression of PTRF by staining a histological sample with PTRF-specific antibodies and the threshold expression level is equal or greater than 3.3. In certain embodiments of this aspect, the threshold is determined for expression of IGFBP7 by determining the protein level in a serum sample and the threshold expression level is equal or greater than 1.4ng/ml. In certain embodiments, the expression is determined for both proteins and compared to the respective threshold.
  • the cancer cell is characterized by a mutation of the BRAF gene, particularly by a BRAFV600E mutation.
  • the method of the invention comprises a step of determining whether a cancer cell representative of the patient’s disease is characterized by a mutation of the BRAF gene, particularly by a BRAFV600E mutation.
  • the cancer cell is a melanoma cell, particularly a metastatic melanoma cell.
  • the level of a marker selected from ALDH1 A1 , PRDX2, PDLIM, OXCT1 , FAM129A, SERPINB6, ALDH1A3, NAMPT, KYNU, UCHL1 , NID1 , POLCE and THBS2 is determined, particularly at least two, more particularly three, four, five or all of ALDH1A1 , PRDX2, PDLIM, OXCT1 , FAM129A, SERPINB6, ALDH1A3, NAMPT, KYNU, UCHL1 , NID1 , POLCE and THBS2 are determined.
  • the level of a marker selected from ALDH, kynurenine and tenascin is determined.
  • the sweat of a fingerprint accessible via a non-invasive and easy procedure, contains several thousand different endogenous and exogenous molecules and thus a lot of molecular information regarding individuals.
  • UHPLC ultra-high performance liquid chromatography
  • Thermo Q Exactive HF fast high-resolution mass spectrometry
  • femtogram amounts of these molecules can be successfully detected and quantified.
  • the inventors have already established the methods to analyse various amino acids and derivatives thereof in finger sweat samples, focussing on tryptophan metabolites such as serotonin, melatonin, kynurenine and others due to their important biological implications.
  • markers identified herein can be determined by new technologies such as (mass spectroscopy based) fast single reaction monitoring (SRM) methods and/or fingerprint methods for metabolites of the tryptophan pathway.
  • SRM fast single reaction monitoring
  • RNA chips are helpful to identify the candidates on protein level and RNA chips of patient biopsies of the candidates on RNA level.
  • the threshold for MAPKi responders is 2.2., and for MAPKI non-responders is 3.3 in the case of PTRF staining.
  • the inventors identified 1 ng/ml in serum samples as threshold for MAPKi responders and 1 4ng/ml for MAPKI non-responders.
  • Another aspect of the invention relates to the use of a MAPK inhibitor in the therapy of cancer characterized by a BRAF mutation, particularly a BRAFV600E mutation.
  • the cancer is malignant melanoma, more particularly metastatic malignant melanoma.
  • the patient is characterized by one or more of the following:
  • the cancer expresses PTRF below a threshold as determined by staining a histological sample with PTRF-specific antibodies at an expression level equal or smaller than 2.2 and / or
  • the patient shows IGFBP7 protein as determined in a serum sample at equal or smaller than 1 ng/ml.
  • the MAPK inhibitor is selected from the group consisting of BRAF inhibitors, particularly sorafenib, vemurafenib, dabrafenib and encorafenib, and MEK inhibitors, particularly trametinib and cobimetinib.
  • Another aspect of the invention relates to the use of an immune checkpoint inhibitor agent or an immune agonist agent, particularly an antibody against PD-1 or PD-L1 , for use in the therapy of cancer characterized by a BRAF mutation a BRAFV600E mutation.
  • the cancer is malignant melanoma, more particularly metastatic malignant melanoma.
  • the patient is characterized by one or more of the following: a. the cancer expresses PTRF above a threshold as determined by staining a histological sample with PTRF-specific antibodies at an expression level equal or greater than 3, particularly > 3.3 and / or
  • the patient shows IGFBP7 protein as determined in a serum sample at equal or greater than 1.4 ng/ml.
  • the invention further relates to a diagnostic kit comprising
  • an ELISA kit for determining IGFBP7 in a serum sample can be constructed, for example, as a sandwich ELISA; this embodiment provides for a vessel, for example a microwell plate, coated with an antibody against IGFBP7, and a second antibody against IGFBP7 provided in solution.
  • the second, soluble antibody can be covalently linked to an enzymatic function allowing detection of the antibody (and thereby the analyte,
  • IGFBP7 by colorimetric assay, luminescence or other methods known in the art, or a secondary antibody reactive to the Fc part of the second IGFBP7 antibody is provided, and this secondary antibody in turn comprises the detectable label, e.g. colormetric or
  • Another aspect of the invention relates to a system for practicing a method according to any one of the aspects or items laid out herein.
  • the system is laid out to determine the susceptibility of a cancer cell to MAPK inhibitor treatment.
  • the expression of the markers identified in the present invention facilitates the identification of patients likely to prove to be responding well to MAPKi therapy. These patients in a next step patients can be transferred to the specific therapy. Good responders having a low expression of the listed markers will be stratified to MAPKi treatment (BRAFi+ MEKi treatment). Patients identified as not having a high probability to respond well to MPAKi therapy are identified prior to initiating treatment and can be stratified to the next best clinical treatment, particularly checkpoint inhibitor therapy targeting the PD-1/PD-1 L interaction, such as anti-PD1 antibodies (immune therapy).
  • Fig. 1 shows that In vivo derived melanoma cultures are resistant to BRAFi
  • A IC50 to PLX4032 and LGX818 of S1 , S2, and R1 -4.
  • B cell adhesion assay of S1 , S2 and R1 -4, ECM-mediated cell adhesion was quantified at OD 560nm after extraction.
  • Fig. 2 shows clinical relevance of PTRF and IGFBP7 and drug activity.
  • A Statistical analysis of immunohistochemical staining for PTRF and based on PFS. * p- value ⁇ 0.05; ** ⁇ 0.01 , *** ⁇ 0.001 ;
  • B Kaplan Meier curves with hazard ratio correlating PFS and TTD (time to death) with PTRF expression.
  • C IGFBP7 analysis in the serum samples of patients with MAPKi therapy by ELISA.
  • Fig. 3 Verification of informative candidates on RNA level DeltaDeltaCT values are depicted in different melanoma cells blue: sensitive cells (S), red: resistant cells (R), light red: intrinsic resistant cells (IR), orange: acquired resistance (AQ)
  • Fig. 4 Hierarchical clustering of the most informative proteins. Proteins needed to distinguish between sensitive and resistant cells in (A) cytoplasm., (B) secretome and (C) nucleus with bootstrap analysis, of the 15 candidates in cell pellets D. Heatmap of the 15 candidates with RNAseq data of S1 , S2 R1 -4 and a second cohort, E, Bootstrap analysis with AU value of 1 st and 2nd proteome and RNA cohort versus proteome signature (15 proteins) and RNA signature of the Verfaillie publication, multiparameter correction.
  • melanoma cell culture M000921 (S1 ) was transduced with a Lentivirus containing a PTRF overexpressing construct (CMV_PTRF) or a non CMV control plasmid (noCMV_PTRF). Expression of PTRF was confirmed by Western blotting. Influence on sensitivity. B, Influence on invasiveness C, Cell adhesion is enhanced in CMV_PTRF in comparison to noCMV_PTRF.
  • Example 1 Patient-derived BRAFi resistant melanoma cultures have an EMT phenotype
  • S1 , S2 Two metastatic primary melanoma cells (S1 , S2) sensitive to the BRAFi LGX818 (Encorafenib) and PLX4032 (Vemurafenib), and four BRAFi-resistant primary cells (R1 , R2, R3, R4), were characterized used for shotgun proteomics analysis.
  • the two cells S1 and S2 were established from skin metastases of treatment naive patients. Four resistant cells were taken from different patients who received BRAFi and progressed under treatment.
  • Resistant melanoma cultures R2 and R3 were established shortly before treatment initiation, suggesting that resistant melanoma cells were already present in these tumors (e.g., intrinsic resistance).
  • the other resistant cultures (R1 and R4) were established from relapsed tumors after an initially successful BRAFi treatment (acquired resistance).
  • R1 and R4 were shown by single-cell cloning and following Sanger sequencing to be double mutated for BRAF V600E and NRAS Q61K or NRAS Q61H , respectively.
  • the IC50 to PLX4032 was the lowest in S2, followed by S1 , R2, R4, R3 and R1 with the highest IC50.
  • the IC50 to LGX818 was the lowest in S2 followed by S1 , R3, R1 , R2 and R4 with the highest IC50 (Fig 1A).
  • the IC50 to MEK162 was highest for R1 (Table below).
  • induced drug-resistant melanoma cells show an increased cell adherence to extracellular matrix proteins.
  • the patient-derived drug-resistant melanoma compared to sensitive cells have a higher adherence mainly to collagen I (p-value: 0.0016) and IV (p-value: 0.022) and fibrinogen (p-value: 0.0274) in a cell adhesion assay (Fig 1 B).
  • the inventors Comparing the sensitive (S1 ) and resistant (R1 ) melanoma cells by zymography, the inventors detected an increase in MMP2 (matrix metalloproteinase 2) activity in the resistant cells (R1 ), a feature associated with invasiveness in EMT. Consistently, N-cadherin levels were increased in culture R1.
  • Example 2 Shotgun proteomic analysis of primary MAPKi sensitive and resistant cells
  • the inventors enriched for subcellular fractions of all cells and performed shotgun proteomics analysis on every fraction. In total, 4052 (unique 1726) proteins in the cytoplasmic, 1007 (unique 81 ) proteins in the supernatant, and 3463 (unique 1328) proteins in the nuclear fraction were detected. Using Perseus software the inventors generated heatmaps for every fraction analyzed and compared protein expression of sensitive versus resistant cells. Unsupervised hierarchical clustering of Z-scored protein abundances by comparing sensitive versus resistant cells of the cytoplasmic fraction yielded comparable results to the nuclear fraction. Few functional classes were found to be upregulated in the secretome of the sensitive and resistant cells.
  • IGFBP7 Insulin-like Growth Factor Binding Protein 7
  • POLCE Procollagen C-endopeptidase enhancer 1
  • NAMPT Nicotinamide phosphoribosyltransferase
  • NID1 Nidogen-1
  • THBS2 Thrombospondin-2
  • IL8 Interleukin 8
  • MITF the master-regulator of melanocyte fate and pigmentation is expressed at a lower level in all resistant cells, highlighting the loss of lineage-specific features associated with drug resistance and suggesting that resistant cells may switch to a mesenchymal phenotype upon resistance.
  • KEGG Knowles's Endothelial migration
  • the inventors enhanced the cohort of primary cells by using cell pellets of the same cohort to confirm the data, integrating relevant new features such as BRAFi and MEKi double resistant primary melanoma cells and intrinsically double resistant melanoma cells in order to analyse MAPKi (BRAFi and MEKi) resistance and to analyse for comparability.
  • This second dataset was composed of 20 primary melanoma cell cultures with 7 MAPKi sensitive primary cells, 4 BRAF resistant equivalent to the first cohort, 5 MAPKi resistant including a model with increasing in vitro derived MEKi resistance and 3 MAPKi intrinsic resistant primary cells. All IC50 values to LGX, PLX and MEK162 and the mutational status to BRAF and NRAS were determined in order of classification (Table). Here cell pellets were generated and MS conducted in order to generalize the findings. This second data set confirmed the upregulation of the pathways found in the first cohort.
  • GSEA Gene Set Enrichment Analysis
  • Example 4 BRAFi and MEKi resistance in KEGG pathway analysis
  • the inventors enhanced the cohort of primary cells by using cell pellets of additional patients of the same cohort to confirm the data, integrating relevant new features such as BRAFi and MEKi double resistant primary melanoma cells and intrinsically double resistant melanoma cells, in order to analyse MAPKi (BRAFi and MEKi) resistance.
  • This second dataset was comprised of 20 primary melanoma cell cultures with 7 MAPKi sensitive primary cells, 4 BRAFi resistant, 5 MAPKi resistant including a cell line with increasing in vitro derived MEKi resistance, and 3 MAPKi intrinsically resistant primary cells. All IC50 values to LGX, PLX and MEK162 and the mutational status of BRAF and NRAS were determined prior to analysis. Cell pellets were made and the MS analysis was conducted at a different institute in order to confirm the earlier findings in a different experimental setting.
  • Example 5 Resistant cells upreaulate translation initiation factors and have a significantly lower pH than sensitive cells
  • EIFs eukaryotic translation initiation factors
  • the proteome data were analysed for possible different drug elimination strategies such as ABCD transporter and V type proton ATPases.
  • ABCD transporter a cluster of proteins of a transporter family (V type proton ATPase) were significantly upregulated in the resistant melanoma cells.
  • V type proton ATPases transport H+ across membranes, for example from the cytoplasm into endosomes.
  • a pH-sensitive fluorescent dye pH-sensitive fluorescent dye (pHrodo dye, Molecular Probes), which fluoresces brightly in acidic environments, was used. Indeed, resistant cell lines showed a significantly stronger fluorescence indicating a lower pH in their endosomes. Therefore, the inventors detected different mechanisms of resistance by proteomics.
  • Example 6 Fifteen proteins have the highest class-discriminatory power between sensitive and resistant cells
  • IGFBP7 IGFBP7 and PTRF (Fig. 4C). Both were among the most strongly differentially expressed proteins of the cytoplasmic fraction. IGFBP7 which is a secreted protein, was also among the candidates that best separated the secretome of drug-sensitive and resistant cells. Therefore, these two proteins were informative for separating sensitive from resistant cells in all analysed fractions and were used for further evaluation in clinical samples.
  • the top 15 proteins that distinguished the two phenotypes were also validated using RNAseq data and were highly differentially expressed in sensitive and resistant cells.
  • RNAseq data of 6 BRAFi and MEKi sensitive and 10 resistant melanoma cells was analysed independently. A clustering with these 15 candidates between both cohorts could be performed for most of the samples.
  • heatmap separation with increasing numbers of proteins sorted by log2FC were generated. Heatmaps for each fraction with 50 proteins are shown depicting that the separation of sensitive and resistant cells remains true for the cytoplasm and for the nuclear fraction; whereas in the secretome, this separation can only be found with the five proteins IGFBP7, NID1 , POLCE, NAMPT and THBS2. Here the heterogeneity is the highest. In addition, in the cytoplasm and nuclear fractions, up- and downregulated proteins were apparently balanced, but the secretome contained the highest proportion of upregulated proteins, as visualized in the GSEA plots and heatmaps of each cellular fraction.
  • candidate proteins were selected using classification to nearest centroids and PTRF, IGFBP7, OXCT1 , PCOLCE and NID1 were also found to be highly informative. Therefore 2 different bioinformatics analyses led to the confirmation of the signature.
  • candidate proteins were selected using classification to nearest centroids and PTRF, IGFBP7, OXCT1 , PCOLCE and NID1 were also found to be highly informative.
  • RNA expression of the top eight proteins that best separated between drug sensitive and resistant cell phenotypes e.g., PTRF, IGFBP7, ALDH1A1 , ALDH1A3, UCHL1 , POLCE, KYNU and NID1 ) was analyzed by RT-PCR.
  • the inventors also included the proinflammatory protein IL-1 b since it was differentially regulated in the resistant cells.
  • MM1 1 1031 (BRAF V600E mutated, intrinsic resistant (IR)) cells, which were isolated from an untreated patient with an aggressive clinical history and a PFS of under 3 months, the in vitro induced drug resistant cell WM983BR (AR / BRAF V600E mutation), and three additional MAPKi drug-resistant cells M140906 (DR-1 , V600K), M140307 (DR-2, BRAF V600E / NRAS Q61R mutation) and M150423 (DR-3, BRAF V600E , NRAS Q61K ).
  • DR1 -3 cells were not only resistant to BRAFi but are also resistant to MEKi and ERKi (Fig 3).
  • PTRF, KYNU, NID1 and IL-1 b appeared to be increased possibly due to the additional resistance to MEK and ERK inhibitors (Fig 3). Overall, all candidate genes were shown to be on average more highly expressed in the resistant cells, although not every resistant cell had high expression of all candidates (Fig 3).
  • the signature for the heatmap was selected using the listed candidates of Verfaillie et. al where a core subset of invasive and proliferative gene signatures was used and additional important genes characterized as main players of the phenotype switch and resistance by Verfaillie et. al, which we call the Verfaillie signature. With these candidates we were also able to clearly separate the RNAseq data from sensitive versus resistant cell cultures indicating that the inventors’ data are comparable to those generated by other groups. In a next step the inventors analysed the two proteome cohorts for the Verfaillie signature and all identified and significant detected proteins of the proteome data was correlated to the phenotypes.
  • the genes correlated to the invasive phenotype are significantly upregulated in the resistant cells; whereas, the genes correlated to the proliferative phenotype (i.e., CDH1 , RAB27A, MLANA, SLC45A2, GPR143, RAB38, SIRPA, MICAL1 , CDK2, MY01 D, NR4A3, MITF, SOX10) are significantly downregulated in the resistant cells. No identified and significantly regulated protein was revealed to be reverse regulated.
  • the inventors performed bootstrap analysis of the 15 protein signature and the Verfaillie signature on both of their proteome cohorts and the RNAseq datasets. This revealed that the inventors were able to verify signatures on RNA or proteome level but the proteome signature has the highest AU p-value for the proteome signature and the RNAseq data for the Verfaillie signature. Therefore, different signatures can be identified by transcriptomics or proteomics, but both technologies are able to verify known signatures indicating the complementary power of both technologies. In addition, both technologies suggested the same cell cultures were outliers (MM130604, MM140307) (Fig 4E).
  • PTRF and IGFBP7 were the most discriminating factors between sensitive and resistant cells.
  • BRAFi and MEKi sensitive melanoma cell cultures have a lower expression of N-cadherin but higher expression of E-cadherin in comparison to the resistant cell cultures.
  • the inventors performed a meta-analysis of microarray data of six international centers where data were publicly available and analyzed for proliferative and invasive melanoma expression signatures (Widmer et al., 2013, The Journal of investigative dermatology, 133(10):2436-43).
  • the inventors found PTRF and IGFBP7 to be significantly upregulated in invasive signature melanoma cells across all 6 analyzed datasets.
  • Example 9 2.8 IGFBP7 expression is downstream of PTRF and PTRF regulates the main features of drug resistance
  • PTRF plays a role in rRNA transcription and the formation and stabilization of caveolae and was not previously known to be functionally involved in melanoma progression.
  • IGFBP7 IGFBP7
  • other proteins that were highly differentially expressed between the sensitive and resistant cells.
  • the inventors treated melanoma cells with siRNA against human PTRF (Yi JS et al., 2013, J Proteome Res., 12(2):605-14).
  • expression analysis revealed downregulation of three (IGFBP7, ALDH1 A3, POLCE) out of the eight candidate proteins from the screen, suggesting that these are downstream targets of PTRF (data not shown).
  • PTRF seems to also be involved in inflammatory processes, since IL-1 b was also down regulated.
  • CRISPR/Cas-derived knockout of PTRF led to the identification of 12 targets which were significantly and at least 2 fold down regulated.
  • These proteins are involved in diverse functions such as immune regulatory processes, cell adhesion, cell migration, angiogenesis, endosomal trafficking, endocytosis, the pH regulation and lysosomal function and IGF signalling.
  • PRKCDBP, TGFBI, EMILIN1 , EHD2, TNC, NGFR, HLA-DRA, NRP2, GPC6, TYMS, IGF2R and TFRC are significantly down regulated.
  • PRKCDBP, TGFBI, EHD2, TNC, and TYMS revealed to be upregulated in all analyzed BRAFi and MEKi resistant cohorts.
  • STRING database analysis showed an indirect link of PTRF and IGFBP7 which can be correlated to the JUN pathway.
  • the phenotype-switching model, as well as the model of EMT in epithelial cancers postulate that invasiveness is induced by factors released from the microenvironment.
  • Tumor- associated fibroblasts are suspected to be a source for such factors (e.g. T ⁇ Rb).
  • T ⁇ Rb Tumor- associated fibroblasts
  • the inventors performed invasion assays using melanoma cells S1 and R1 with and without conditioned media from tumor- associated fibroblasts. Both melanoma cells invade matrigel and transmigrate through the chamber membrane, whereby the resistant cells (R1 ) show this phenotype to a significantly higher extent.
  • R1_APTRF CRISPR/Cas
  • Beta-catenin was previously reported to be localized at the membrane in proliferative melanoma cells while invasive melanoma cells show diffuse cytoplasmic localization.
  • the inventors can confirm that resistant R1 cells showing fibroblastic cell features do have cytoplasmic beta-catenin staining. Deleting PTRF resulted in changes in the cell morphology with observed membranous localization of beta-catenin similar to sensitive S1 melanoma cells suggesting that PTRF is partly responsible for beta-catenin localization in focal contacts as well as Caveolin-1 expression and localization.
  • PTRF is highly upregulated (green circle) and the ALDH1 A1 and A3 proteins. Performing nearest shrunken centroid revealed that only ALDH1A3 is necessary to separate both cohorts (red circle). Using DAVID bioinformatic software i.e.“cell cell adhesion” or “regulation of apoptotic process” were revealed to be upregulated in CMV_PTRF.
  • Example 10 PTRF and IGFBP7 expression are sipnificant biomarkers for BRAFi therapy
  • IGFBP7 was the only other protein needed in the nucleus for predictive power and was the most differentially expressed protein. Since it is a secreted protein, the inventors performed an ELISA of 19 serum samples of patients receiving MAPKi therapy, and serum samples from healthy donors as controls. The inventors could confirm significantly higher IGFBP7 levels in the blood of patients after relapse (Fig 2C). Therefore, the marker might as well work as pharmacodynamics marker.
  • the inventors performed an ELISA of 19 serum samples of patients receiving BRAFi or BRAFi and MEKi combination therapy, and serum samples from healthy donors as controls. They could confirm significantly higher IGFBP7 levels in the blood of patients with PFS under 3 months (Fig 2 C).
  • Healthy donors have the same expression level than patients which are good responders which suggests that the expression level is not correlated with tumor mass. If this threshold is exceeded low responders can be identified.
  • the threshold always belongs to one group:
  • PTRF up to 2.2 good responder this refers to the staining of melanoma metastasis of patients >9month PFS with the mentioned ranking criteria
  • 3.3 and/or nuclear staining bad responder to MAPKi this refers to the staining of melanoma metastasis of patients ⁇ 3month PFS with the mentioned ranking criteria
  • IGFBP7 everything under 1 ng/ml good responder (measured in serum samples of patients with good response or healthy donors by ELISA), over 1 .4ng/ml bad responder to MAPKi responder (measured in serum samples of patients with bad response by ELISA).
  • Example 11 Proteomics profiles identify a drug effective in BRAFi-resistant melanoma cells
  • the inventors used pathway analysis to elucidate pathways upregulated in the drug-resistant melanoma cell signatures.
  • One pathway that was highly differentially expressed was the lysosomal pathway.
  • the endo-lysosomal pathway has recently been shown by gene set enrichment analysis (GSEA) to be specifically prominent in melanoma compared to more than 35 other cancer types (Alonso-Curbelo et al. Cancer Cell 2014).
  • GSEA gene set enrichment analysis
  • melanoma cells were established from patient biopsies using the selective adherence method (Raaijmakers Ml et al., 2015, Experimental dermatology, 24(5):377-80). Cells were grown in RPMI 1640 (Sigma Life Science, USA) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, USA), 2mM glutamine (Biochrom, Germany) and sodium pyruvate (Sigma Life Science, USA).
  • the BRAFi Vemurafenib (PLX4032), Encorafenib (LGX818) and MEKi Binimetinib (MEK162) were purchased at Selleckchem, USA.
  • the ERK1/2 inhibitor SCH772984 was purchased at Chemical probes. All used primary cells and the IC50 are listed in Table EV 2.
  • Melanoma cells were seeded to a density of 2.5 x 10 3 cells in each well of a 96-well-plate and challenged with dose-escalating concentrations of BRAFi for 72h.
  • Cell viability was estimated using a colorimetric (MTT) assay. Briefly, on the day of the assay the medium was changed to medium containing 10% MTT-stock solution (stock solution 10 mg/ml MTT in PBS, Sigma- Aldrich, Switzerland). Plates were incubated at 37 °C for 2 to 4 hours depending on the cell line, medium was removed and formazan crystals were solubilized in 100 pi dimethyl sulfoxide (Sigma- Aldrich, Switzerland). Absorbance was measured at 595 nm (reference 620 nm) using a microplate reader (Tecan infinite M200 Pro). The half-maximal inhibitory concentration (IC50 value) was calculated using GraphPad Prism software (USA).
  • Melanoma cells (S1 and R1 ) were pre-starved with RPMI containing reduced FBS concentration (3%) for 48h. On the day of the experiment 2x10 4 melanoma associated fibroblast were seeded in 800 mI in 24-well plates and let to adhere. Empty and matrigel-coated invasion inserts (8 pm pore size, BD Biosciences, Bedford, Massachusetts) were re-hydrated in RPMI without FBS for 2 hours. Pre-starved melanoma cells were trypsinzed and collected into RPMI without FBS. Cell titer was adjusted to 1 x10 5 cells/ml.
  • ECM Array 48-Well Cell Adhesion Assay
  • melanoma cells were seeded in a density of 1 x10 5 cells/well in ECM proteins pre coated 24-well plates and incubated for 1 hour at 37 °C. Unbound cells were washed away and the adherent cells were fixed in 70% ethanol and stained with 0.2% crystal blue staining solution and quantified colorimetrically.
  • Blocks of paraffin-embedded, formalin-fixed tissues corresponding to melanoma patients who received targeted therapy were used for immunohistochemical analysis, which was performed as described in Belloni et al. (Belloni B et al., 2012, Chemical immunology and allergy, 97:191 - 202).
  • the following primary antibodies were used: rabbit polyclonal Anti-PTRF antibody (ab48824, Abeam) (1 :200), monoclonal mouse Anti- CD3 (DAKO M7254) (1 :50), mouse monoclonal anti-CD99 (ab17083, Abeam) (1 :10) and rabbit monoclonal anti-MHC class I antibody (ABIN650045).
  • the zymography assay was performed as described in Paulitschke et al. (2012, PloS one, 7(9):e46103. Epub 2012/10/1 1 ). The supernatants of the primary cells were collected. The SDS gel contained gelatine (1 mg/ml), was stained in Coomassie solution for 30 minutes and stripped with an isopropanol-acetic acid solution (BioTeZ Berlin-Buch GmbH, Berlin, Germany).
  • cytoplasmic fraction the primary melanoma cells were lysed in isotonic lysis buffer (10 mM HEPES/NaOH, pH 7.4, 0.25 M sucrose, 10 mM NaCI, 3 mM MgCI2, 0.5% T riton X-100) supplemented with protease inhibitors (pepstatin, leupeptin and aprotinin, each at 1 pg/ml; 1 mM PMSF) and mechanical shear stress.
  • protease inhibitors pepstatin, leupeptin and aprotinin, each at 1 pg/ml; 1 mM PMSF
  • nuclear fractionation By centrifugation at 2300xg at 4°C the cytoplasmic proteins were separated from the nuclei and precipitated overnight with ice-cold ethanol at -20°C.
  • cell pellets were used for the second cohort. Supernatant was collected after 6 hours incubation in serum free medium and sterile filtered through a 0.2
  • sample buffer 7.5 M urea, 1.5 M thiourea, 4% CHAPS, 0.05% SDS, 100 mM DDT
  • protein concentrations were determined by means of a Bradford assay (Bio-Rad-Laboratories, Germany).
  • Peptides of the cellular fractions were separated using nanoflow UHPLC (Thermo Easy-nLC 1000) using a 15 cm x 75 pm PepMap RSLC C18 analytical column (2 pm particle size, Thermo Fisher Scientific). Peptide separation was achieved applying a linear gradient of 5% to 30% mobile phase B (98% ACN, 2% H2O, 0.15% FA; mobile phase A contained 98% H2O, 2% ACN, 0.15% FA) over 180 min at a flow rate of 300 nl/min. Peptides were analyzed in positive ionization mode using ion trap CID in an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) in data-dependent acquisition mode.
  • search criteria included a maximum of two missed cleavages, a minimum of two peptide identifications per protein, at least one of them unique, and an FDR less than 0.01 for both peptide identification as well as protein identification.
  • carbamidomethylation of cysteine residues was set as a fixed modification and oxidation of methionine residues and N-terminal protein acetylation as variable modifications.
  • the LFQ-values as these are Log2 values, the difference directly corresponds to logarithmic fold-control values.
  • Protein identifications were further analyzed using Perseus (version 1 .3.0.4). TProteins were filtered for reversed sequences and contaminants as well as a minimum of three independent identifications per protein. Significantly up- and down-regulated proteins were determined by applying a two-sided t-test with a significance level of p ⁇ 0.05 (permutation-based correction). In addition, hierarchical clustering was achieved using Euclidean distance and average linkage clustering of Z-scored expression values.
  • GSEA Gene Set Enrichment Analysis
  • GSEA Gene Set Enrichment Analysis
  • a classifier based on shrunken centroids was constructed using ClaNC and R (Dabney AR et al., 2006, Bioinformatics, 22(1 ):122-3). Priors were chosen according to the number of samples. The performance of the classifier was tested by leaving-one-out cross validation.
  • Heatmaps were constructed using standard R procedures, hierarchical clustering and the Pearson correlation coefficient with average linkage as distance metrics. Different sizes of heatmaps were performed.
  • siRNA transfection of melanoma cells was carried out using INTERFERE transfection solution according to the manufacturer’s protocol (Polyplus-transfection, France). Cells were transfected with 20 nM of siRNA (Qiagen) for 72 h before RNA or protein was extracted. A PTRF specific antisense knock-down mRNA was employed. Non-human transcript targeting anti-sense RNA was used as a negative control (AllStars negative control, Qiagen, Netherlands).
  • CRISPR CRISPRed cell cultures of MM121224 were generated using the MuLE (Multiple Lentiviral Expression) System according to Albers et al (Albers J et al., 2015, J Clin Invest,
  • sgRNAs used in this study were designed according to the CRISPR Design Tool (https://chopchop.rc.fas.harvard.edu/) as standard primers (Microsynth) and depicted in Table EV 1 .
  • the PAM sequence depicted in blue was not included in the cloned sgRNA.
  • a 5’ ACCG was added to the 5’ end of the forward direction oligo and a 5’ AAAC to the 5’ end of the reverse complement oligo.
  • Oligos were first annealed to generate double stranded DNA fragments and then ligated into the BfuAI-digested Entry vector (pMuLE ENTR U6 stuffer sgRNA scaffold L1 -R5 Plasmid #62127, Addgene). The sequence of the resulting Entry vectors was verified by Sanger sequencing.
  • the Entry vector (Plasmid #62127, Addgene) containing the gRNA, the Entry vector containing Cas9 (pMuLE ENTR SV40-hCas9 L5-L2 Plasmid #62134, Addgene) and a lentiviral destination vector (expressing EGFP) were recombined using the Gateway LR Clonase II Plus Enzyme mix (Life Technologies #12538-120).
  • Lentivirus was prepared as described recently (81 ). HEK293T cell supernatant was centrifuged at 1 .200 g for 5 min and then applied on subconfluent melanoma cells.
  • Cells positive for GFP were sorted by FACS using an ARIA3 device. Bulk culture was amplified followed by single-cell sorting also using the ARIA3 device. Cell clones were tested for the absence of the target protein by Western Blot and in case of PTRF were further analyzed by proteomics.
  • RNA-Seq RNA-Seq Stranded mRNA protocol and sequenced on the lllumina HiSeq2500 paired end 125 bp.
  • RNA-Seq data RNA-Seq data were aligned using the R implementation of subread with Hg19 as a reference genome (Liao Y et al., 2013, Nucleic acids research, 41 (10):e10).. Mapped reads were summarized on the gene level. Genes with less than 5 reads over all samples were filtered previous to analysis. Differential expression was estimated using DEGSeq2 (Love Ml et al., 2014, Genome biology, 15(12):550). For visualization in heatmaps, the z-score of the moderated logarithm of normalized counts was used.
  • SEM with S1 , S2, R1 -R4 was performed as described in Paulitschke et al. and Bray et al. (Paulitschke V et al., 2015, Molecular cancer therapeutics, Epub 2015/01/24; Bray DF et al., 1993, Microsc Res Tech, 26(6):489-95, Epub 1993/12/15).
  • the SEM images were taken at a magnification between 500 and 3000 using a Leo DSM 982 field emission scanning electron microscope at 4 kV.
  • pH-sensitive staining cells were seeded as above, stained according to manufacturer’s instruction (pHrodo® dye, Molecular Probes®) and imaged using identical exposure and acquisition conditions. Relative fluorescence of single cells in each image was quantified using ImageJ software.
  • Sphere formation of cultured cells require no-adhesive conditions in cell culture dishes.
  • the inventors added 100 mI of RPMI containing 5.000 cells to each well and let the cells incubate at 37°C for 48h.
  • Calcein AM Sigma-Aldrich
  • Ethidium homodimers ThermoFischer
  • caveolin-1 expression sequesters beta- catenin at the membrane during the course of progression or that that absence of PTEN lead to caveolin-1 dependent beta-catenin transcription regulation (Lobos-Gonzalez et al. Pigment cell & melanoma research. 2013;26(4):555-70, Conde-Perez et al., Nature communications. 2015;6:8093).
  • the inventors have found that upon induction of PTRF expression there was no alteration in the localization of beta-catenin.
  • the BRAFmutated and drug sensitive melanoma cell culture M000921 (S1 ) was transduced with Lentivirus contains PTRF overexpressing construct (CMV_PTRF) or a non CMV control plasmid (noCMV_PTRF). Expression of PTRF was confirmed by Western blotting (A). Cells were subjected to dose-escalating concentrations of the BRAFi LGX818 and viability was measured. Fluorescent microscopy revealed coregulation of PTRF and Caveolin-1. Rapid spheroid formation is observed in PTRF expressing melanoma cultures which also show higher degree of invasion into a collagen 1 matrix compared to cell cultures lacking PTRF expression).
  • PTRF functions as a regulator of sensitivity and invasion in BRAF mutated melanoma and acts therefore as functional marker.
  • PTRF lentiviral expressing constructs were generated using the multiple lentiviral expression sytem (MuLE) as described by Albers et al., 2015 (Albers and Ian Frew, University Zurich). Shortly, PTRF was cloned from a commercially available vector (Origene SC101318) into either the ENTRY vector pMuLE ENTR CMV L5-L2 (Addgene 62091 ) or as control into the ENTRY vector pMuLE ENTR MCS L1 -R5 (Addgene 61084).
  • Lentivirus particles were prepared via transfection of subconfluent HEK293T cells cultured in DMEM with 10% FCS.
  • 8 pg of total vector DNA containing CMV_PTRF_eGFP or noCMV_PTRF_eGFP vector and the lentiviral packaging plasmids psPAX2 (Addgene, no 12260) and pMD2.G (Addgene, no 12259)) were mixed in a ratio 4:2:1 in 1 ml of serum-free DMEM.
  • 24 mI PEI (1 mg/ml) was added and incubated for 15 minutes at RT and mixed with 9 ml DMEM with 10% FCS before mixture was added to HEK cells.
  • Supernatant containing lentivirus particles were harvested twice in 96h, spin at 1500g for 10 minutes and melanoma cells were incubated with supernatant for 72h before sorted for GFP expression with an Aria III 4L cell sorter.
  • Cluster uncertainty was estimated using pvclust, which is an implementation of multiscale bootstrap resampling for assessing the uncertainty in hierarchical cluster analysis. It provides AU (approximately unbiased) p-value as well as BP (bootstrap probability) value for each cluster in a dendrogram.
  • AU p-value which is computed by multiscale bootstrap resampling, is a better approximation to unbiased p-value than BP value computed by normal bootstrap resampling, therefore this value is used http://www.sigmath.es.osaka-u.ac.jp/shimo- lab/prog/pvclust/.
  • a 96-well plate was coated with 100 mI of 1 .5% Agarose dissolved in RPMI containing no supplements or FCS and kept under UV light in the laminar flow until agarose polymerized. 5000 cells per well were seeded on top of the agarose solution in 100 mI melanoma cell culture medium. Spheroid formation was followed over several days using a Zeiss Primovert light microscope.
  • Spheroids were also embedded in Collagen 1 (from rat tails, BD, USA). Here spheres were harvested and kept on ice. Residual cell culture medium was removed and sphere was mixed with 100 mI Collagen 1 solution containing 10% FCS, 10% DMEM, 4 mM L-Glutamine and 0.4 % Sodiumbicarbonate and transferred into a 96-well plate coated with 1.5 Agarose. Collagen solution and let to polymerize in the cell culture incubator for 1 h. Collagen was topped up100 mI of melanoma medium.
  • Table Category of primary cells and the IC50 to MAPKi treatment.
  • Items 1. A method of assigning to a cancer patient a likelihood of being responsive to treatment with a MAPK inhibitor, wherein the method comprises the steps of
  • cancer cell sample is characterized by a mutation of the BRAF gene, particularly by a BRAFV600E mutation.
  • PTRF by staining a histological sample with PTRF-specific antibodies and the threshold expression level is equal or smaller than 2.5, particularly ⁇ 2.2 and / or b.
  • IGFBP7 by determining the protein level in a serum sample and the threshold
  • expression level is equal or smaller than 1 ng/ml.
  • a method for assigning a patient suffering from a cancer to treatment with an immune checkpoint inhibitor agent comprising the steps of
  • PTRF by staining a histological sample with PTRF-specific antibodies and the threshold expression level is equal or greater than 3, particularly >3.3 and / or b.
  • IGFBP7 by determining the protein level in a serum sample and the threshold
  • expression level is equal or greater than 1.4ng/ml.
  • said cancer cell is a melanoma cell, particularly a metastatic melanoma cell.
  • the expression level of a marker selected from ALDH1 A1 , PRDX2, PDLIM, OXCT 1 , FAM129A, SERPINB6, ALDH1A3, NAMPT, KYNU, UCHL1 , NID1 , POLCE and THBS2 is determined, particularly at least two, more particularly three, four, five or all of ALDH1A1 , PRDX2, PDLIM, OXCT1 , FAM129A, SERPINB6, ALDH1A3, NAMPT, KYNU, UCHL1 , NID1 , POLCE and THBS2 are determined.
  • the cancer expresses PTRF below a threshold as determined by staining a
  • the patient shows IGFBP7 protein as determined in a serum sample at equal or smaller than 1 ng/ml.
  • MAPK inhibitor for use in the therapy of cancer according item 10, wherein said MAPK inhibitor is selected from the group consisting of BRAF inhibitors, particularly sorafenib, vemurafenib, dabrafenib and encorafenib, and MEK inhibitors, particularly trametinib and cobimetinib.
  • BRAF inhibitors particularly sorafenib, vemurafenib, dabrafenib and encorafenib
  • MEK inhibitors particularly trametinib and cobimetinib.
  • An immune checkpoint inhibitor agent for use in the therapy of cancer characterized by a BRAF mutation, particularly malignant melanoma, more particularly metastatic malignant melanoma, wherein the patient is characterized by one or more of the following:
  • the cancer expresses PTRF above a threshold as determined by staining a
  • the patient shows IGFBP7 protein as determined in a serum sample at equal or greater than 1.4 ng/ml.
  • a diagnostic kit comprising
  • an antibody to PTRF wherein the antibody is fluorescently labelled
  • an ELISA kit for determining IGFBP7 in a serum sample a. an antibody to PTRF, wherein the antibody is fluorescently labelled

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Abstract

L'invention concerne un procédé d'attribution d'un patient souffrant d'un cancer à un traitement avec un agent inhibiteur de point de contrôle immunitaire, consistant à déterminer dans un échantillon de cellules cancéreuses obtenu à partir du patient un niveau d'expression d'un biomarqueur choisi parmi PTRF et IGFBP7, à comparer ledit niveau d'expression à un seuil et à attribuer audit patient un traitement immédiat avec un agent inhibiteur de point de contrôle immunitaire si ledit niveau d'expression est inférieur audit seuil.
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