WO2013155077A1 - Response markers for src inhibitor therapies - Google Patents
Response markers for src inhibitor therapies Download PDFInfo
- Publication number
- WO2013155077A1 WO2013155077A1 PCT/US2013/035783 US2013035783W WO2013155077A1 WO 2013155077 A1 WO2013155077 A1 WO 2013155077A1 US 2013035783 W US2013035783 W US 2013035783W WO 2013155077 A1 WO2013155077 A1 WO 2013155077A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- braf
- cancer
- subject
- src
- cells
- Prior art date
Links
- 238000002560 therapeutic procedure Methods 0.000 title claims abstract description 46
- 229940122924 Src inhibitor Drugs 0.000 title claims abstract description 43
- 230000004044 response Effects 0.000 title claims description 16
- 101000984753 Homo sapiens Serine/threonine-protein kinase B-raf Proteins 0.000 claims abstract description 171
- 102100027103 Serine/threonine-protein kinase B-raf Human genes 0.000 claims abstract description 170
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 102
- 230000000694 effects Effects 0.000 claims abstract description 75
- 238000000034 method Methods 0.000 claims abstract description 66
- 201000011510 cancer Diseases 0.000 claims abstract description 63
- 230000002829 reductive effect Effects 0.000 claims abstract description 38
- 108010091528 Proto-Oncogene Proteins B-raf Proteins 0.000 claims abstract description 23
- 102000018471 Proto-Oncogene Proteins B-raf Human genes 0.000 claims abstract description 23
- 238000012360 testing method Methods 0.000 claims abstract description 12
- 239000002067 L01XE06 - Dasatinib Substances 0.000 claims description 144
- 229960002448 dasatinib Drugs 0.000 claims description 144
- ZBNZXTGUTAYRHI-UHFFFAOYSA-N Dasatinib Chemical compound C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1Cl ZBNZXTGUTAYRHI-UHFFFAOYSA-N 0.000 claims description 139
- 102100033479 RAF proto-oncogene serine/threonine-protein kinase Human genes 0.000 claims description 99
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 71
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims description 71
- 108091000080 Phosphotransferase Proteins 0.000 claims description 62
- 102000020233 phosphotransferase Human genes 0.000 claims description 62
- 108090000623 proteins and genes Proteins 0.000 claims description 32
- 239000000523 sample Substances 0.000 claims description 30
- 102000001332 SRC Human genes 0.000 claims description 29
- 101150048834 braF gene Proteins 0.000 claims description 29
- 102000004169 proteins and genes Human genes 0.000 claims description 24
- 239000003112 inhibitor Substances 0.000 claims description 20
- 238000011282 treatment Methods 0.000 claims description 17
- 229940043355 kinase inhibitor Drugs 0.000 claims description 15
- 239000003757 phosphotransferase inhibitor Substances 0.000 claims description 15
- 108010087686 src-Family Kinases Proteins 0.000 claims description 14
- 230000008859 change Effects 0.000 claims description 12
- 208000020816 lung neoplasm Diseases 0.000 claims description 11
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 10
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims description 10
- 201000005202 lung cancer Diseases 0.000 claims description 10
- 230000004083 survival effect Effects 0.000 claims description 10
- OUKYUETWWIPKQR-UHFFFAOYSA-N saracatinib Chemical compound C1CN(C)CCN1CCOC1=CC(OC2CCOCC2)=C(C(NC=2C(=CC=C3OCOC3=2)Cl)=NC=N2)C2=C1 OUKYUETWWIPKQR-UHFFFAOYSA-N 0.000 claims description 9
- 229950009919 saracatinib Drugs 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- 229960003862 vemurafenib Drugs 0.000 claims description 7
- GPXBXXGIAQBQNI-UHFFFAOYSA-N vemurafenib Chemical group CCCS(=O)(=O)NC1=CC=C(F)C(C(=O)C=2C3=CC(=CN=C3NC=2)C=2C=CC(Cl)=CC=2)=C1F GPXBXXGIAQBQNI-UHFFFAOYSA-N 0.000 claims description 7
- MLDQJTXFUGDVEO-UHFFFAOYSA-N BAY-43-9006 Chemical compound C1=NC(C(=O)NC)=CC(OC=2C=CC(NC(=O)NC=3C=C(C(Cl)=CC=3)C(F)(F)F)=CC=2)=C1 MLDQJTXFUGDVEO-UHFFFAOYSA-N 0.000 claims description 6
- 239000005511 L01XE05 - Sorafenib Substances 0.000 claims description 6
- 239000002145 L01XE14 - Bosutinib Substances 0.000 claims description 6
- 206010027476 Metastases Diseases 0.000 claims description 6
- UBPYILGKFZZVDX-UHFFFAOYSA-N bosutinib Chemical group C1=C(Cl)C(OC)=CC(NC=2C3=CC(OC)=C(OCCCN4CCN(C)CC4)C=C3N=CC=2C#N)=C1Cl UBPYILGKFZZVDX-UHFFFAOYSA-N 0.000 claims description 6
- 229960003736 bosutinib Drugs 0.000 claims description 6
- 230000009401 metastasis Effects 0.000 claims description 6
- 229960003787 sorafenib Drugs 0.000 claims description 6
- 238000006467 substitution reaction Methods 0.000 claims description 6
- 239000002177 L01XE27 - Ibrutinib Substances 0.000 claims description 5
- XKFTZKGMDDZMJI-HSZRJFAPSA-N N-[5-[(2R)-2-methoxy-1-oxo-2-phenylethyl]-4,6-dihydro-1H-pyrrolo[3,4-c]pyrazol-3-yl]-4-(4-methyl-1-piperazinyl)benzamide Chemical compound O=C([C@H](OC)C=1C=CC=CC=1)N(CC=12)CC=1NN=C2NC(=O)C(C=C1)=CC=C1N1CCN(C)CC1 XKFTZKGMDDZMJI-HSZRJFAPSA-N 0.000 claims description 5
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims description 5
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 claims description 5
- 229950002966 danusertib Drugs 0.000 claims description 5
- 229960001507 ibrutinib Drugs 0.000 claims description 5
- XYFPWWZEPKGCCK-GOSISDBHSA-N ibrutinib Chemical compound C1=2C(N)=NC=NC=2N([C@H]2CN(CCC2)C(=O)C=C)N=C1C(C=C1)=CC=C1OC1=CC=CC=C1 XYFPWWZEPKGCCK-GOSISDBHSA-N 0.000 claims description 5
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims description 5
- 229960001285 quercetin Drugs 0.000 claims description 5
- 235000005875 quercetin Nutrition 0.000 claims description 5
- HUNGUWOZPQBXGX-UHFFFAOYSA-N tirbanibulin Chemical compound C=1C=CC=CC=1CNC(=O)CC(N=C1)=CC=C1C(C=C1)=CC=C1OCCN1CCOCC1 HUNGUWOZPQBXGX-UHFFFAOYSA-N 0.000 claims description 5
- 238000011319 anticancer therapy Methods 0.000 claims description 4
- 239000012472 biological sample Substances 0.000 claims description 4
- 238000013500 data storage Methods 0.000 claims description 4
- 238000012217 deletion Methods 0.000 claims description 4
- 230000037430 deletion Effects 0.000 claims description 4
- 238000000338 in vitro Methods 0.000 claims description 4
- 206010050017 Lung cancer metastatic Diseases 0.000 claims description 3
- 206010058019 Cancer Pain Diseases 0.000 claims description 2
- 102000004127 Cytokines Human genes 0.000 claims description 2
- 108090000695 Cytokines Proteins 0.000 claims description 2
- 238000011122 anti-angiogenic therapy Methods 0.000 claims description 2
- 238000002512 chemotherapy Methods 0.000 claims description 2
- 229960002465 dabrafenib Drugs 0.000 claims description 2
- BFSMGDJOXZAERB-UHFFFAOYSA-N dabrafenib Chemical compound S1C(C(C)(C)C)=NC(C=2C(=C(NS(=O)(=O)C=3C(=CC=CC=3F)F)C=CC=2)F)=C1C1=CC=NC(N)=N1 BFSMGDJOXZAERB-UHFFFAOYSA-N 0.000 claims description 2
- 238000001415 gene therapy Methods 0.000 claims description 2
- 238000001794 hormone therapy Methods 0.000 claims description 2
- 230000007170 pathology Effects 0.000 claims description 2
- 238000001959 radiotherapy Methods 0.000 claims description 2
- 238000001356 surgical procedure Methods 0.000 claims description 2
- 208000024891 symptom Diseases 0.000 claims description 2
- 230000004614 tumor growth Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- ZCCPLJOKGAACRT-UHFFFAOYSA-N 4-methyl-3-[[1-methyl-6-(3-pyridinyl)-4-pyrazolo[3,4-d]pyrimidinyl]amino]-N-[3-(trifluoromethyl)phenyl]benzamide Chemical compound CC1=CC=C(C(=O)NC=2C=C(C=CC=2)C(F)(F)F)C=C1NC(C=1C=NN(C)C=1N=1)=NC=1C1=CC=CN=C1 ZCCPLJOKGAACRT-UHFFFAOYSA-N 0.000 claims 3
- 150000004922 Dasatinib derivatives Chemical group 0.000 claims 3
- 210000004027 cell Anatomy 0.000 description 173
- 230000035772 mutation Effects 0.000 description 88
- 101100523539 Mus musculus Raf1 gene Proteins 0.000 description 67
- 230000000415 inactivating effect Effects 0.000 description 35
- 230000035945 sensitivity Effects 0.000 description 31
- 230000009758 senescence Effects 0.000 description 30
- 102100031968 Ephrin type-B receptor 2 Human genes 0.000 description 28
- 108010007457 Extracellular Signal-Regulated MAP Kinases Proteins 0.000 description 27
- 230000004913 activation Effects 0.000 description 24
- 210000001519 tissue Anatomy 0.000 description 21
- 230000037361 pathway Effects 0.000 description 18
- 230000003213 activating effect Effects 0.000 description 17
- 230000001771 impaired effect Effects 0.000 description 17
- 108060006706 SRC Proteins 0.000 description 16
- 238000004458 analytical method Methods 0.000 description 16
- 230000014509 gene expression Effects 0.000 description 16
- 102000004232 Mitogen-Activated Protein Kinase Kinases Human genes 0.000 description 15
- 108090000744 Mitogen-Activated Protein Kinase Kinases Proteins 0.000 description 15
- 238000001262 western blot Methods 0.000 description 15
- 238000006471 dimerization reaction Methods 0.000 description 14
- 201000001441 melanoma Diseases 0.000 description 14
- 108700024394 Exon Proteins 0.000 description 13
- 108020004459 Small interfering RNA Proteins 0.000 description 13
- -1 VP-BHG712 Chemical compound 0.000 description 13
- 230000005764 inhibitory process Effects 0.000 description 13
- 230000006907 apoptotic process Effects 0.000 description 12
- 231100000002 MTT assay Toxicity 0.000 description 11
- 238000000134 MTT assay Methods 0.000 description 11
- 108020004707 nucleic acids Proteins 0.000 description 11
- 102000039446 nucleic acids Human genes 0.000 description 11
- 102000015098 Tumor Suppressor Protein p53 Human genes 0.000 description 10
- 108010078814 Tumor Suppressor Protein p53 Proteins 0.000 description 10
- 230000006870 function Effects 0.000 description 10
- 238000003752 polymerase chain reaction Methods 0.000 description 10
- 238000010186 staining Methods 0.000 description 10
- 102100030708 GTPase KRas Human genes 0.000 description 9
- 101000584612 Homo sapiens GTPase KRas Proteins 0.000 description 9
- 238000003197 gene knockdown Methods 0.000 description 9
- 238000001114 immunoprecipitation Methods 0.000 description 9
- 102000016914 ras Proteins Human genes 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 102000005936 beta-Galactosidase Human genes 0.000 description 8
- 108010005774 beta-Galactosidase Proteins 0.000 description 8
- 230000007246 mechanism Effects 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- 230000035899 viability Effects 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 230000002441 reversible effect Effects 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 108010009392 Cyclin-Dependent Kinase Inhibitor p16 Proteins 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 101100221606 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) COS7 gene Proteins 0.000 description 6
- 102100033254 Tumor suppressor ARF Human genes 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 230000026969 oncogene-induced senescence Effects 0.000 description 6
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 5
- 229940125431 BRAF inhibitor Drugs 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 102100036723 Discoidin domain-containing receptor 2 Human genes 0.000 description 5
- 101710127786 Discoidin domain-containing receptor 2 Proteins 0.000 description 5
- 102100031480 Dual specificity mitogen-activated protein kinase kinase 1 Human genes 0.000 description 5
- 101710146526 Dual specificity mitogen-activated protein kinase kinase 1 Proteins 0.000 description 5
- 102000001301 EGF receptor Human genes 0.000 description 5
- 108060006698 EGF receptor Proteins 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 238000002591 computed tomography Methods 0.000 description 5
- 231100000135 cytotoxicity Toxicity 0.000 description 5
- 230000003013 cytotoxicity Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 230000002018 overexpression Effects 0.000 description 5
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 4
- 230000003350 DNA copy number gain Effects 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 206010061289 metastatic neoplasm Diseases 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 230000026731 phosphorylation Effects 0.000 description 4
- 238000006366 phosphorylation reaction Methods 0.000 description 4
- 230000008707 rearrangement Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000002269 spontaneous effect Effects 0.000 description 4
- 206010041823 squamous cell carcinoma Diseases 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 3
- 108700012813 7-aminoactinomycin D Proteins 0.000 description 3
- 101150016325 EPHA3 gene Proteins 0.000 description 3
- 102100030340 Ephrin type-A receptor 2 Human genes 0.000 description 3
- 102100030324 Ephrin type-A receptor 3 Human genes 0.000 description 3
- 102100036725 Epithelial discoidin domain-containing receptor 1 Human genes 0.000 description 3
- 101710131668 Epithelial discoidin domain-containing receptor 1 Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101001009087 Homo sapiens Tyrosine-protein kinase HCK Proteins 0.000 description 3
- 108010055717 JNK Mitogen-Activated Protein Kinases Proteins 0.000 description 3
- 206010069755 K-ras gene mutation Diseases 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 101150038994 PDGFRA gene Proteins 0.000 description 3
- 108010014608 Proto-Oncogene Proteins c-kit Proteins 0.000 description 3
- 102000016971 Proto-Oncogene Proteins c-kit Human genes 0.000 description 3
- 102100027389 Tyrosine-protein kinase HCK Human genes 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 230000022131 cell cycle Effects 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 230000004077 genetic alteration Effects 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 238000007901 in situ hybridization Methods 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 238000000021 kinase assay Methods 0.000 description 3
- 231100000225 lethality Toxicity 0.000 description 3
- 230000001394 metastastic effect Effects 0.000 description 3
- 230000000869 mutational effect Effects 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000007480 sanger sequencing Methods 0.000 description 3
- 238000002741 site-directed mutagenesis Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 2
- 101150023956 ALK gene Proteins 0.000 description 2
- 102000000872 ATM Human genes 0.000 description 2
- 239000012110 Alexa Fluor 594 Substances 0.000 description 2
- 108010004586 Ataxia Telangiectasia Mutated Proteins Proteins 0.000 description 2
- 101100220616 Caenorhabditis elegans chk-2 gene Proteins 0.000 description 2
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 2
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 2
- 102100022732 Diacylglycerol kinase beta Human genes 0.000 description 2
- 108010055196 EphA2 Receptor Proteins 0.000 description 2
- 102000009465 Growth Factor Receptors Human genes 0.000 description 2
- 108010009202 Growth Factor Receptors Proteins 0.000 description 2
- 101001044814 Homo sapiens Diacylglycerol kinase beta Proteins 0.000 description 2
- 101001128135 Homo sapiens NACHT, LRR and PYD domains-containing protein 4 Proteins 0.000 description 2
- 101000702545 Homo sapiens Transcription activator BRG1 Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 239000005536 L01XE08 - Nilotinib Substances 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 2
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 2
- 102100031898 NACHT, LRR and PYD domains-containing protein 4 Human genes 0.000 description 2
- 102000043276 Oncogene Human genes 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 206010033701 Papillary thyroid cancer Diseases 0.000 description 2
- 108010001267 Protein Subunits Proteins 0.000 description 2
- 102000002067 Protein Subunits Human genes 0.000 description 2
- 206010056342 Pulmonary mass Diseases 0.000 description 2
- 101150040459 RAS gene Proteins 0.000 description 2
- 101150076031 RAS1 gene Proteins 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 208000006265 Renal cell carcinoma Diseases 0.000 description 2
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 2
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 102100031027 Transcription activator BRG1 Human genes 0.000 description 2
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 2
- 239000000061 acid fraction Substances 0.000 description 2
- 208000009956 adenocarcinoma Diseases 0.000 description 2
- 208000036878 aneuploidy Diseases 0.000 description 2
- 231100001075 aneuploidy Toxicity 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000005754 cellular signaling Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000002648 combination therapy Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000000710 homodimer Substances 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000010820 immunofluorescence microscopy Methods 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 201000002037 lung adenoma Diseases 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229960001346 nilotinib Drugs 0.000 description 2
- HHZIURLSWUIHRB-UHFFFAOYSA-N nilotinib Chemical compound C1=NC(C)=CN1C1=CC(NC(=O)C=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)=CC(C(F)(F)F)=C1 HHZIURLSWUIHRB-UHFFFAOYSA-N 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 238000011338 personalized therapy Methods 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 208000037821 progressive disease Diseases 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 239000012723 sample buffer Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000004055 small Interfering RNA Substances 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 208000030045 thyroid gland papillary carcinoma Diseases 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- KKVYYGGCHJGEFJ-UHFFFAOYSA-N 1-n-(4-chlorophenyl)-6-methyl-5-n-[3-(7h-purin-6-yl)pyridin-2-yl]isoquinoline-1,5-diamine Chemical compound N=1C=CC2=C(NC=3C(=CC=CN=3)C=3C=4N=CNC=4N=CN=3)C(C)=CC=C2C=1NC1=CC=C(Cl)C=C1 KKVYYGGCHJGEFJ-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 102100033793 ALK tyrosine kinase receptor Human genes 0.000 description 1
- 102100022900 Actin, cytoplasmic 1 Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 102100036409 Activated CDC42 kinase 1 Human genes 0.000 description 1
- 102100032599 Adhesion G protein-coupled receptor B3 Human genes 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000269350 Anura Species 0.000 description 1
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 102100027515 Baculoviral IAP repeat-containing protein 6 Human genes 0.000 description 1
- 101150017888 Bcl2 gene Proteins 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010005949 Bone cancer Diseases 0.000 description 1
- 208000018084 Bone neoplasm Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 102100039683 Cyclin-G-associated kinase Human genes 0.000 description 1
- 101150062301 Ddr2 gene Proteins 0.000 description 1
- 101001053785 Drosophila melanogaster Dual specificity mitogen-activated protein kinase kinase dSOR1 Proteins 0.000 description 1
- 102100040067 E3 ubiquitin-protein ligase TRIM36 Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101150076616 EPHA2 gene Proteins 0.000 description 1
- 101150097734 EPHB2 gene Proteins 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108010055211 EphA1 Receptor Proteins 0.000 description 1
- 101150078651 Epha4 gene Proteins 0.000 description 1
- 102100030322 Ephrin type-A receptor 1 Human genes 0.000 description 1
- 102100021616 Ephrin type-A receptor 4 Human genes 0.000 description 1
- 102100030779 Ephrin type-B receptor 1 Human genes 0.000 description 1
- 102100031982 Ephrin type-B receptor 3 Human genes 0.000 description 1
- 108091006010 FLAG-tagged proteins Proteins 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 101710113436 GTPase KRas Proteins 0.000 description 1
- 102100039788 GTPase NRas Human genes 0.000 description 1
- 241000287826 Gallus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 102100031188 Hephaestin Human genes 0.000 description 1
- 108010034791 Heterochromatin Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000928956 Homo sapiens Activated CDC42 kinase 1 Proteins 0.000 description 1
- 101000796801 Homo sapiens Adhesion G protein-coupled receptor B3 Proteins 0.000 description 1
- 101000936081 Homo sapiens Baculoviral IAP repeat-containing protein 6 Proteins 0.000 description 1
- 101000886209 Homo sapiens Cyclin-G-associated kinase Proteins 0.000 description 1
- 101000610402 Homo sapiens E3 ubiquitin-protein ligase TRIM36 Proteins 0.000 description 1
- 101001064150 Homo sapiens Ephrin type-B receptor 1 Proteins 0.000 description 1
- 101001064458 Homo sapiens Ephrin type-B receptor 3 Proteins 0.000 description 1
- 101000744505 Homo sapiens GTPase NRas Proteins 0.000 description 1
- 101000993183 Homo sapiens Hephaestin Proteins 0.000 description 1
- 101001131830 Homo sapiens PDZ domain-containing RING finger protein 4 Proteins 0.000 description 1
- 101000605639 Homo sapiens Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Proteins 0.000 description 1
- 101000692455 Homo sapiens Platelet-derived growth factor receptor beta Proteins 0.000 description 1
- 101000606502 Homo sapiens Protein-tyrosine kinase 6 Proteins 0.000 description 1
- 101000694802 Homo sapiens Receptor-type tyrosine-protein phosphatase T Proteins 0.000 description 1
- 101000591205 Homo sapiens Receptor-type tyrosine-protein phosphatase mu Proteins 0.000 description 1
- 101000823271 Homo sapiens Tyrosine-protein kinase ABL2 Proteins 0.000 description 1
- 101000912503 Homo sapiens Tyrosine-protein kinase Fgr Proteins 0.000 description 1
- 101001022129 Homo sapiens Tyrosine-protein kinase Fyn Proteins 0.000 description 1
- 101001054878 Homo sapiens Tyrosine-protein kinase Lyn Proteins 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 108091092878 Microsatellite Proteins 0.000 description 1
- 101100381978 Mus musculus Braf gene Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102100029166 NT-3 growth factor receptor Human genes 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102100034575 PDZ domain-containing RING finger protein 4 Human genes 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 241000233805 Phoenix Species 0.000 description 1
- 102100038332 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Human genes 0.000 description 1
- 102100026547 Platelet-derived growth factor receptor beta Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 102100039810 Protein-tyrosine kinase 6 Human genes 0.000 description 1
- 241001510071 Pyrrhocoridae Species 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 102100028645 Receptor-type tyrosine-protein phosphatase T Human genes 0.000 description 1
- 102100034090 Receptor-type tyrosine-protein phosphatase mu Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 208000007660 Residual Neoplasm Diseases 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 102000038012 SFKs Human genes 0.000 description 1
- 108091008118 SFKs Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 102100031463 Serine/threonine-protein kinase PLK1 Human genes 0.000 description 1
- 208000024313 Testicular Neoplasms Diseases 0.000 description 1
- 206010057644 Testis cancer Diseases 0.000 description 1
- 206010051259 Therapy naive Diseases 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 102100022651 Tyrosine-protein kinase ABL2 Human genes 0.000 description 1
- 102100026150 Tyrosine-protein kinase Fgr Human genes 0.000 description 1
- 102100035221 Tyrosine-protein kinase Fyn Human genes 0.000 description 1
- 102100026857 Tyrosine-protein kinase Lyn Human genes 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 229940124650 anti-cancer therapies Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000005773 cancer-related death Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 230000025084 cell cycle arrest Effects 0.000 description 1
- 238000003783 cell cycle assay Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 239000004568 cement Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000012303 cytoplasmic staining Methods 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000013501 data transformation Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 229950007919 egtazic acid Drugs 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 210000004458 heterochromatin Anatomy 0.000 description 1
- 238000005734 heterodimerization reaction Methods 0.000 description 1
- 102000050152 human BRAF Human genes 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 239000012133 immunoprecipitate Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 208000037841 lung tumor Diseases 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 108010056274 polo-like kinase 1 Proteins 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 238000002600 positron emission tomography Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 238000012342 propidium iodide staining Methods 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 102000009929 raf Kinases Human genes 0.000 description 1
- 108010077182 raf Kinases Proteins 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- CMZUMMUJMWNLFH-UHFFFAOYSA-N sodium metavanadate Chemical compound [Na+].[O-][V](=O)=O CMZUMMUJMWNLFH-UHFFFAOYSA-N 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 102000009076 src-Family Kinases Human genes 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000009121 systemic therapy Methods 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 201000003120 testicular cancer Diseases 0.000 description 1
- 239000003656 tris buffered saline Substances 0.000 description 1
- 108010064892 trkC Receptor Proteins 0.000 description 1
- 238000013042 tunel staining Methods 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 229910000166 zirconium phosphate Inorganic materials 0.000 description 1
Classifications
-
- 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
-
- 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
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- 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
- A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
- A61K31/437—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
-
- 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
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
-
- 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
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
-
- 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
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
-
- 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
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- 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
- G01N33/57423—Specifically defined cancers of lung
-
- 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
- G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
-
- 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
- C12Q2600/156—Polymorphic or mutational 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 generally to the field of molecular biology and oncology. More particularly, it concerns methods for treating cancer and methods for predicting a response to anti-cancer therapies.
- NSCLC non-small cell lung cancer
- EGFR epidermal growth factor receptor
- EML4-ALK translocations for which treatment with targeted agents has produced profound clinical responses.
- This development has elicited a paradigm shift in treating lung cancer: the tumors' genetic characteristics dictate therapy. Nevertheless, definition of clinically relevant genetic aberrations is lacking in about 85% of NSCLC cases.
- a method for identifying a candidate for a Src inhibitor therapy comprising (a) testing a biological sample from a subject having a cancer to determine whether cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity relative to a wild type BRAF protein; and (b) identifying the subject as a good candidate for a Src inhibitor therapy if the sample from the subject indicates that cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity; or identifying the subject as a poor candidate for a Src inhibitor therapy if the sample from the subject does not indicates that cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity.
- a method for identifying a good candidate for a Src inhibitor therapy comprising testing a biological sample from a subject having a cancer to determine whether cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity relative to a wild type BRAF protein, thereby identifying the subject as a good candidate for a Src inhibitor therapy.
- testing the sample in accordance with the embodiments comprises measuring BRAF kinase activity in the sample.
- testing a sample comprises determining whether cells of the cancer comprises a BRAF gene encoding a protein with reduced kinase activity.
- the BRAF gene can encode a protein comprising an amino acid substitution or deletion at position Y472 (e.g., a Y472C amino acid substitution).
- testing a sample comprises determining all or part of a BRAF gene sequence in the sample.
- a sample is a blood sample, a tissue sample, a lymph sample or a saliva sample.
- a good candidate for a Src inhibitor therapy can be, without limitation, a subject that is predicted to have reduced in tumor size or burden, reduced tumor growth, reduced tumor-associated pain, reduced cancer associated pathology, reduced cancer associated symptoms, cancer non-progression, cancer remission, reduced cancer metastasis, or increased survival time in response to a Src inhibitor therapy.
- a method of the embodiments comprises providing a report, such as a written or electronic report, indicating whether the subject is a good candidate or a poor candidate for a Src inhibitor therapy.
- a report is transmitted to a third party such as a hospital, a doctor, an insurance company or a healthcare provider.
- a method for treating a subject having a cancer comprising administering a Src inhibitor therapy (e.g., a Src kinase inhibitor) to the subject.
- a method can comprise administering a Src inhibitor therapy 1, 2, 3, 4, 5, 6 or more times to the subject over a period of days, weeks or months.
- a method of the embodiments further comprises administering at least a second anti-cancer therapy to the subject, such as chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy, anti- angiogenic therapy or cytokine therapy.
- a composition is provided for the treatment of a subject having cancer comprising cancer cells with reduced BRAF kinase activity relative to a control level, the composition comprising a therapeutically effective amount of a Src inhibitor.
- a method for treating a subject having a cancer comprising administering to the subject an effective amount of a Src inhibitor in conjunction with (e.g., before, after or concurrently with) a RAF inhibitor.
- the Src inhibitor can be a Src kinase inhibitor, such as bosutinib, saracatinib, danusertib, VP-BHG712, quercetin, PCI-32765, KX2-391, AP23451 or dasatinib.
- the RAF inhibitor is a BRAF inhibitor, such as a BRAF specific inhibitor.
- RAF inhibitors include, without limitation, vemurafenib, GSK2118436 and sorafenib.
- a composition is provided for the treatment of a subject having cancer comprising a therapeutically effective amount of a Src inhibitor and a RAF inhibitor.
- a subject of the embodiments is a subject having or diagnosed with a cancer, such as a lung, breast, brain, prostate, spleen, pancreatic, cervical, ovarian, head and neck, esophageal, liver, skin, kidney, leukemia, bone, testicular, colon, or bladder cancer.
- a cancer such as a lung, breast, brain, prostate, spleen, pancreatic, cervical, ovarian, head and neck, esophageal, liver, skin, kidney, leukemia, bone, testicular, colon, or bladder cancer.
- the cancer can be a lung cancer such as a non-small cell or a metastatic lung cancer.
- a Src inhibitor therapy of the embodiments is a Src kinase inhibitor.
- the Src kinase inhibitor can be bosutinib, saracatinib, danusertib, VP-BHG712, quercetin, PCI-32765, KX2-391, AP23451 or dasatinib.
- a computer that, when executed by a computer, causes the computer to perform operations comprising (a) receiving information corresponding to a BRAF kinase activity in a sample from a subject having a cancer; and (b) determining a relative level of BRAF kinase activity compared to a reference level, wherein reduced BRAF kinase activity relative to the reference level indicates the subject is a good candidate for a Src inhibitor therapy.
- the information corresponding to a BRAF kinase activity can comprises all or part of a BRAF gene sequence.
- determining a relative level of BRAF kinase activity comprises determining a change in all or part of a BRAF gene sequence compared to a reference sequence, wherein a change in the BRAF gene sequence corresponding to reduced BRAF activity indicates that the subject is a good candidate for a Src inhibitor therapy.
- a reference level of the embodiments can be all or part of a wild type BRAF gene sequence or all or part of a BRAF gene sequence corresponding to protein with increased or decreased kinase activity relative to a wild type sequence.
- receiving information in accordance with the embodiments comprises receiving from a tangible data storage device information corresponding to a level of BRAF kinase activity in a sample from a subject having a cancer.
- a tangible computer-readable medium of the embodiments further comprises a computer-readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising: sending information corresponding to the relative level of BRAF kinase activity to a tangible data storage device.
- a tangible computer-readable medium comprises a computer-readable code that, when executed by a computer, causes the computer to perform operations further comprising (c) calculating a diagnostic score for the sample, wherein the diagnostic score is indicative of the probability that the subject will respond to a Src inhibitor therapy (e.g., whether the subject is a good candidate for a Src inhibitor therapy).
- FIG. 1 Y472C BRAF is biochemically similar to G466V BRAF but different from 6 00E BRAF.
- IP immunoprecipitation
- MEK kinase-dead MEK (substrate) and ATP
- MEK phosphorylation was measured using Western blotting and quantitated with ImageJ.
- B-D The effect of the expression of mutated BRAF proteins on MEK and ERK activity in intact cells. Western blotting was performed in (B) COS7, (C) H661, and (D) H226 cells transfected with BRAF constructs.
- FIG. 2 NSCLC cells with an inactivating BRAF mutation undergo senescence when exposed to dasatinib.
- A-D Biological effects of dasatinib in NSCLC cells with BRAF mutations. NSCLC cells were incubated with 150 nM dasatinib or a control vehicle for 72 hours followed by ⁇ -galactosidase (A) staining and (B) quantification, (C) cell cycle analysis using propidium iodide staining and FACS analysis, or (D) BrdU incorporation.
- A staining and
- B quantification
- C cell cycle analysis using propidium iodide staining and FACS analysis
- D BrdU incorporation.
- E The effect of dasatinib on downstream signaling as measured by Western blotting of NSCLC cells incubated with 150nM dasatinib for 24 hours.
- FIG. 3 Expression of kinase inactive and active BRAF leads to sensitivity and resistance to dasatinib, respectively, in NSCLC cells.
- A, B, D Cell viability was assayed following dasatinib treatment in NSCLC cells transfected with BRAF constructs.
- FIG. 4 Dasatinib indirectly inhibits CRAF activity in cells with an inactivating BRAF mutation but not in cells with ⁇ BRAF or 600E BRAF.
- CRAF knock down (KD) The effect of CRAF knock down (KD) on NSCLC cell number in cells with endogenous BRAF mutations. Untransfected Catl2T, H1666, H322, and H661 cells were incubated with CRAF siRNA, and their viability was estimated using an MTT assay. In all cases, Western blotting was used to confirm CRAF knockdown. At 120h Call2T cells lost dasatinib sensitivity and CRAF expression recovered.
- FIG. 5 Dastinib induces RAF dimerization in NSCLC cells which is necessary for sensitivity in cells expressing kinase impaired BRAF.
- A Dasatinib induced RAF dimerization. NSCLC cells were incubated with 150 nM dasatinib for 72 hours then CRAF was immunoprecipitated. The resulting lysate was split with part used for blotting with antibodies to BRAF or CRAF (to measure dimerization) and part used to measure CRAF kinase activity using MEK as a substrate.
- B-C Introduction of a mutation that interferes with RAF dimerization abrogates the effects of kinase deficient BRAF on dasatinib sensitivity.
- FIG. 7. (A) Radiographic images of PX's primary lung tumor and paraspinal metastasis, demonstrating that only a residual lung nodule remains over 3 years after the completion of treatment with dasatinib alone for 12 weeks. (B) Kaplan-Meier overall survival and progression free survival curves for the 34 patients enrolled on the dasatinib phase 2 study at a median of 36 months of follow up.
- FIG. 8 Alignment of the human BRAF (Homo; ; SEQ ID NO:3) amino acid sequence with that of mice (Mus; SEQ ID NO: L>, rats (Rattus; SEQ ID NO:2), chickens (Gallus; SEQ ID NO:4), and frogs (Xenopus; ; SEQ ID NO:5).
- the tyrosine residue marked in red represents the Y472 site in humans, which is conserved in all tested evolutionary distant organisms.
- FIG. 9 NSCLC cells with kinase inactive BRAF mutations did not undergo apoptosis when exposed to 150nM dasatinib for 72 hours as measured by TUNEL staining.
- FIG. 10 Cells with inactive BRAF undergo senescence in the presence of dasatinib. Immunofluorescence microscopy was done with Call2T, HI 666 (A), H661 and H2987 (B) cell lines treated with vehicle or 150 nM dasatinib for 72 hours. Cells were labeled with senescence-associated heterochromatin foci marker HPl- ⁇ and counterstained with DAPI. Induction of HPl- ⁇ in Dasatinib treated cells is significantly higher in Call2-T and HI 666. (C) Fold change in HPl- ⁇ expressing cells in dasatinib treated group compared to vehicle control in three independent experiments.
- FIG. 11 HI 666 cells were incubated with 100 nM dasatinib for the indicated times followed by replacement with fresh media (schedule per left panel) and senescence was quantitated with ⁇ -galactosidase staining using light microscopy. (*P value ⁇ 0.05).
- FIG. 12. H661 cells transfected with inactivating BRAF mutations show increased sensitivity to dasatinib. H661 cells were transfected with the noted BRAF constructs and incubated with increasing doses of dasatinib for 72 hours. Their viability was estimated using an MTT assay. These data were used to generate the data presented in FIG. 3A.
- FIG. 13 Transfection with mutant or wt BRAF does not affect cell number.
- HI 666 and H661 cells were transfected with the noted BRAF constructs and cell number was estimated 72 hours later using the MTT assay.
- FIG. 14 NSCLC cells with an inactivating BRAF mutation are sensitive to CRAF knockdown (KD). H661 cells expressing the noted BRAF proteins were incubated with CRAF siRNA, and their viability was estimated using an MTT assay. In all cases, Western blotting was used to confirm CRAF knockdown.
- FIG. 15 Kinase inhibitor- induced RAF dimerization does not result in drug sensitivity or senescence.
- NSCLC cells were incubated with the noted drugs (dasatinib: 150 nM, nilotinib: 2 ⁇ , bosutinib: 1.5 ⁇ and AZD0530: 5 ⁇ respectively) for 72 hours followed by immuoprecipitation with CRAF from Call2T cells (A), MTT assay (B), or ⁇ - galactosidase staining (C).
- FIG. 16 Dasatinib does not have any BRAF mutation-specific changes in BAD or JNK phosphorylation.
- the noted NSCLC cell lines were incubated with 150 nM dasatinib for 72 hours followed by Western blotting with the noted antibodies.
- FIG. 17 Inhibition of c-Src does not affect cell viability in NSCLC harboring a kinase inactive BRAF mutation. H1666 cells were incubated with AZD0530, c-Src siRNA, or controls.
- a and B Western blotting confirmed c-Src knock down or inhibition .
- C Neither c-Src siRNA nor controls affect cell viability 72 hours following transfection. (note: The MTT assay for AZD0530 is included in FIG. 15B).
- FIG. 18 Dasatinib enhances the cytotoxicity of sorafenib in cancer cells resistant to dasatinib. Dasatinib-resistant head and neck cancer (top four panels) and NSCLC (bottom four panels) cells were incubated with 100 nM dasatinib and 1-10 ⁇ sorafenib for 72 hours, and their viability was estimated using an MTT assay. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
- Metastatic NSCLC is a common and fatal disease with a 4-year survival rate of only 2%, but personalized therapies targeting specific genetic aberrations in NSCLC tumors are remarkably successful.
- a patient was identified with stage IV NSCLC with long term disease control by single-agent dasatinib therapy alone. His tumor harbored a kinase inactivating Y472C BRAF mutation that was likely responsible for its unusual sensitivity to dasatinib. Although effective treatments are available for melanoma patients with activating BRAF mutations, no such therapies are available for cancer patients who harbor inactivating mutations.
- No inactivating BRAF mutations were identified in any other patients in the clinical trial, all of whom were nonresponders.
- NSCLC cell lines with endogenous inactivating BRAF mutation underwent senescence when exposed to dasatinib; the cell lines' dasatinib sensitivity reversed with the overexpression of active BRAF.
- NSCLC cells transfected with an activating BRAF mutation were more resistant to dasatinib than were controls, transfection of NSCLC cells with kinase impaired BRAF led to their increased dasatinib sensitivity.
- BRAF mutations occur in only about 4% of NSCLC cases but are more common in other tumors, such as melanoma (50%) and papillary thyroid cancer (40%) (19,20). The majority of BRAF mutations cause activation of the kinase (20). In NSCLC, 57% of mutations are remaining 43% are a mixture of kinase inactivating, activating, and uncharacterized mutations (19). CRAF mutations are rare in all cancers (21). [0041] Interactions between CRAF and BRAF are complex and incompletely understood despite several recent elegant studies in melanoma (4, 5, 22, 23).
- the pathway is not linear and is further complicated by the multiplicity of signaling molecule components; BRAF and CRAF are similar to each other but do not function identically and are not interchangeable and feedback pathways lead to inactivation of upstream components of the pathway (i.e., a negative feedback amplifier) (24). Additionally, MEK is not the only RAF substrate (25).
- BRAF-CRAF interactions The complexity of BRAF-CRAF interactions is epitomized by the RAF inhibitor paradox. Specifically, inhibition of BRAF activity in cells that express 600E BRAF results in expected inhibition of ERK activity and subsequent apoptosis; melanomas with 6 00E BRAF respond clinically to BRAF inhibition (26). Paradoxically, BRAF inhibition in cells with active RAS leads to ERK activation via activation of CRAF (4). These observations are supported by previous research demonstrating that CRAF and BRAF heterodimers are more active than is either the CRAF or BRAF homodimer even when the BRAF protomer has an inactivating mutation (23).
- Oncogene-induced senescence occurs after activation of oncogenes such as RAS and RAF. Classically, it is mediated by activation of the ⁇ / ⁇ ) and/or pi 4 ARF /p53 tumor suppressor pathway. Senescence also can be induced and mediated by other pathways, such as those involving c-Src, STAT3, c-Myc, FOX04, Chk2, and c-Jun-N-terminal-kinase. In the present study, dasatinib induced senescence in HI 666 and Call2T cells but induced apoptosis in H661 cells into which were transfected kinase impaired BRAF mutations.
- Y472C BRAF functions similarly to other known inactivating BRAF mutations and was likely responsible for PX's response to dasatinib. Moreover, the potential for synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options. Given that dasatinib and BRAF inhibitors are in clinical use, this work has potential for direct clinical applications.
- Example 1 Patient's Tumor Analysis
- PX male former smoker
- CT computed tomography
- PX's tumor did not harbor any EGFR or KRAS mutations by intron-based polymerase chain reaction (PCR) ⁇ -Ras exons 1 and 2 (codons 12, 13, and 61) and EGFR exons 18-21 as previously published (2).
- PCR polymerase chain reaction
- no ALK gene rearrangements were detected by fluorescence in situ hybridization
- no c-Src mutations were detected by intron- based PCR of exons 7-10; nor were any discoidin domain receptor 2 (DDR2) mutations detected using conventional Sanger Sequencing of all exons as previously described (6).
- Immunohistochemistry revealed that the tumor did express total and phosphorylated EphA2, c-Kit, and PDGFRa (Table 1).
- PX did not harbor any germ line BRAF or KRAS mutations by intron-based PCR of BRAF exons 1 1 and 15 and K-Ras exons 1 and 2 (codons 12, 13, and 61) of DNA isolated from his peripheral blood lymphocytes.
- the MassARRAY system Sequenom
- aCGH performed. No mutations were detected among the 40 genes tested (Table 2).
- Using aCGH several regions of increased and decreased copy numbers were detected (Table 3).
- Increased copy numbers of the known direct dasatinib targets HCK, DDR1, EPHA3, and ARG (ABL2) were also detected.
- Table 1 Immunchistochemistry scores for PX's tumor.
- Table 2 Genes analyzed using mass spectroscopy single-nucleotide polymorphism analysis.
- Table 3 Copy number variation for genes associated with dasatinib targeting and/or NSCLC.
- Table 4 Mutational statuses of patients with NSCLC treated with dasatinib.
- Y472C BRAF site-directed mutagenesis was used to create Y472C BRAF, G466 BRAF (kinase-impaired), and 600E BRAF (constitutively active) in a Flag-tagged ⁇ BRAF construct.
- the constructs were transfected into COS7 cells, the Flag-tagged proteins isolated and tested for kinase activity.
- V600E BRAF had increased kinase activity
- G466V BRAF had reduced kinase activity.
- Y472C BRAF showed severely reduced kinase activity that was less than 10% that of ⁇ BRAF (FIG. 1A).
- Example 3 Lung Cancer Cells Expressing Y472C BRAF Express Activated MEK, ERK, and CRAF
- CRAF kinase activity was assessed in H661 cells transfected with the BRAF constructs panel. Cells expressing
- G466V BRAF or Y472C BRAF CRAp actiyity man d j d mQs e ceUs express i ng ⁇ BRAF or (FIG. i ImE).
- ⁇ L ;ik1 ewise, c _eilils express ;ing G466V Bn>nRATF? o flesh carer Y Y 4 4 7 / 2C L T BRAF had higher BRAF-CRAF binding than did cells expressing BRAF or VWJU 3 ⁇ 4RAF (FIG. IF).
- H661 and H226 cells were transfected with the panel of BRAF constructs. Expression of the inactivating mutations increased the cells' sensitivity to dasatinib, whereas expression of 600E BRAF induced further resistance (FIGs. 3A, 3B, and 12). In contrast HI 666 and Call2T cells, which have endogenous BRAF inactivation ( G466 BRAF), H661 cells, transfected with kinase impaired BRAF, underwent apoptosis and not senescence when exposed to dasatinib (FIG. 3C).
- H2087 L597V Activating >5000 64 NRAS, TP53, DGKB, DGKB, PTPRT, ATM, BAI3, HECWl,
- dasatinib induces senescence in NSCLC cells with kinase-deficient BRAF. It was hypothesized that dasatinib induces senescence by affecting CRAF function (8). Although it was found that dasatinib did not directly affect CRAF or BRAF kinase activity at relevant concentrations (FIG. 4A), which is consistent with published studies (7, 9), it led to decreased CRAF activity in intact cells that express kinase inactive BRAF (FIG. 4B). To establish the importance of CRAF in NSCLC cells with kinase impaired BRAF, CRAF expression was knocked-down using siRNA in NSCLC cells with kinase active or inactive BRAF.
- H1666 G466V BRAF
- Call2T GA66 BRAF
- H322 wt BRAF
- H661 wt BRAF
- dasatinib induced RAF dimerization in cells with a KRAS mutation (A549) (10).
- dasatinib induced RAF dimerization in NSCLC cells with kinase impaired BRAF (FIG. 5A).
- dasatinib caused a modest inhibition of RAF kinase activity, when corrected for the increase in total RAF dimers, there was no net inhibition of RAF in Call2T and H1666 cells.
- ERK was activated and p21 expression induced after dasatinib treatment in cells with kinase impaired BRAF consistent with oncogene induced senescence that is observed with KRAS or BRAF activation (FIG. 2E) (1 1).
- ERK and MEK were inhibited in H661 and A549 cells, which is consistent with their lack of senescence following dasatinib exposure.
- Example 7 The Combination of a BRAF Inhibitor and Dasatinib Leads to Synergistic
- Metastatic NSCLC is a common and fatal disease with a 4-year survival rate of only 2%, but personalized therapies targeting specific genetic aberrations in NSCLC tumors are remarkably successful.
- a patient was identified with stage IV NSCLC with long term disease control by single-agent dasatinib therapy alone. His tumor harbored a kinase inactivating Y472C BRAF mutation that was likely responsible for its unusual sensitivity to dasatinib. Although effective treatments are available for melanoma patients with activating BRAF mutations, no such therapies are available for cancer patients who harbor inactivating mutations.
- BRAF mutations occur in only about 4% of NSCLC cases but are more common in other tumors, such as melanoma (50%) and papillary thyroid cancer (40%) (19,20). The majority of BRAF mutations cause activation of the kinase (20). In NSCLC, 57% of mutations are remaining 43% are a mixture of kinase inactivating, activating, and uncharacterized mutations (19). CRAF mutations are rare in all cancers (21).
- BRAF inhibition in cells that express 600E BRAF results in expected inhibition of ERK activity and subsequent apoptosis; melanomas with 6 00E BRAF respond clinically to BRAF inhibition (26).
- BRAF inhibition in cells with active RAS leads to ERK activation via activation of CRAF (4).
- Oncogene-induced senescence occurs after activation of oncogenes such as RAS and RAF. Classically, it is mediated by activation of the p ⁇ /Rb and/or pi 4 ARF /p53 tumor suppressor pathway. Senescence also can be induced and mediated by other pathways, such as those involving c-Src, STAT3, c-Myc, FOX04, Chk2, and c-Jun-N-terminal-kinase. In the present study, dasatinib induced senescence in HI 666 and Call2T cells but induced apoptosis in H661 cells into which were transfected kinase impaired BRAF mutations.
- Antibodies used in this study were phosphorylated SFK (pSFK, Y416), pERK 1/2 (T202/Y204), p21 cip , Bcl2, total ERK, pMEK-1/2 (S217/221), phospho-EphA2 (Tyr594), and p-c-Kit (Y719) (Cell Signaling Technology); total CRAF (BD Biosciences); total BRAF, phospho-platelet-derived growth factor (pPDGFRa; Y754), total PDGFRa, c-Kit, Flag M2, and agarose-conjugated CRAF (Santa Cruz Biotechnology); Flag and ⁇ -actin (Sigma Chemical Co); and p53 (Dako).
- Cytotoxicity in NSCLC cells was assessed using a 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described (31). Under each experimental condition, at least four independent wells were treated. Median effects of drugs on viability were calculated using the Chou-Talalay equation (32) with the CalcuSyn software program (Biosoft).
- NSCLC cells were processed using a senescence-associated ⁇ -galactosdiase staining kit (Cell Signaling Technology) according to the manufacturer's instructions and visualized under an Olympus 1X71 phase microscope (Olympus America).
- Cell Signaling Technology Cell Signaling Technology
- Olympus 1X71 phase microscope Olympus America
- cells were washed with phosphate-buffered saline to remove residual media and fixed.
- a ⁇ -galactosidase staining solution containing X-gal was then added to the fixed cells and incubated at 37°C overnight in a dry incubator without CO 2 . Fields with at least 100 cells were counted, in triplicate.
- FACS fluorescence-activated cell sorter
- TU EL terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling
- BrdU incorporation was measured according to the manufacturer's instruction (BrdU Flow Kits, BD Biosciences). Briefly, subconfluent cell cultures were treated with DMSO or 150 nM dasatinib for 72 hours, then labeled with 10 ⁇ BrdU for 4 hr. Cells were trypsinized, fixed, and stained with FITC- conjugated anti-BrdU antibody and 7-Aminoactinomycin D (7-AAD). Samples were analyzed by two-dimensional flow cytometry to detect both fluorescein and 7-AAD.
- Nucleofector Solution V (Amaxa)
- small interfering RNA siRNA; 200 pmol/100 ⁇
- Cells were then electroporated using the Nucleofector program U24 (Amaxa) and diluted with a prewarmed 500- ⁇ RPMI medium supplemented with 10% serum and plated onto 60-mm plates and the medium was changed 16 hours later.
- the c-Src and CRAF siRNAs were predesigned as sets of four independent sequences (siGENOME SMARTpool; Dharmacon). Controls were cells transfected with a nontargeting (scrambled) siRNA and mock-transfected cells (i.e., no siRNA).
- Flag-tagged ⁇ BRAF and 600E BRAF plasmids were provided by Dr. Walter Kolch (Systems Biology Ireland and The Conway Institute, University College Dublin).
- the ⁇ BRAF construct was used as a template to create the Y472C BRAF , G466 BRAF, R509H BRAF and TM TM ⁇ BRAF mutations using the QuikChange XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions.
- the sense mutagenic primers used were 5'-GATCATTTGGAACAGTCTGCAAGGGAAAGTGGCATGGT -3' (SEQ ID NO: 6) for Y412C BRAF and 5'-GTGGGACAAAGAATTGGATCTGTATCATTTGGAACA GTC -3' (SEQ ID NO: 7) for G466W BRAF, 5'-
- IP immunoprecipitation
- the immunoprecipitates were washed four times with an immunocomplex wash buffer (50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 1% Triton X-100, 1 mmol/L egtazic acid, 1 mmol/L ethylenediaminetetraacetic acid, 1% glycerol, 20 ⁇ g/mL leupeptin, 10 ⁇ g/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium vanadate) and boiled with l x sample buffer for 5 min.
- an immunocomplex wash buffer 50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 1% Triton X-100, 1 mmol/L egtazic acid, 1 mmol/L ethylenediaminetetraacetic acid, 1% glycerol,
- gDNA from the specimens was analyzed using polymerase chain reaction (PCR) amplification followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectroscopy single-nucleotide polymorphism analysis (MassARRAY; Sequenom, Inc., San Diego, CA) for 40 genes with 240 mutations (Table 1).
- PCR products were classified as wt or mutant according to their molecular weights. Detected mutations were then Sanger sequenced.
- the primers used for c-Src were CTT CTC CTT TCC TCC CTC CTT (forward; SEQ ID NO:9) and CAG GAG AGG CAC TCT GCAC (reverse; SEQ ID NO: 10) for exon 7, AGC CAT ATC CAG GGA GAA GC (forward; SEQ ID NO: l 1) and ACA CCC AGC TCA AAC CAC TC (reverse; SEQ ID NO: 12) for exon 8, CCT TCC CTC CAA TGT CAG G (forward; SEQ ID NO: 13) and AGT CTG CAG CTG AGG CTT TG (reverse; SEQ ID NO: 14) for exon 9, and AGA AGA CCC GCC TAA CTG CT (forward; SEQ ID NO: 15) and ATC CAG CAG AGG CAG CTA AAG (reverse; SEQ ID NO: 16) for exon 10.
- the primers used for BRAF were TCC CTC TCA GGC ATA AGG TAA (forward; SEQ ID NO: 17) and CGA ACA GTG AAT ATT TCC TTT GAT (reverse; SEQ ID NO: 18) for exon 11 and TCA TAA TGC TTG CTC TGA TAG GA (forward; SEQ ID NO: 19) and GGC CAA AAA TTT AAT CAG TGG A (reverse; SEQ ID NO:20) for exon 15. Mutational Analysis of BRAF and c-Src
- Intron-based PCR was used to sequence B-Raf exons 1 1 and 15 and c-Src exons 7-10 as described previously.
- FFPE tumor tissue was microdissected, and about 200 cells were used in each amplification.
- DNA extracted from either microdissected tissue or cell pellets was subjected to PCR, and PCR products were directly sequenced using a PRISM dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). The primers are listed in the supplemental methods. Sequence variants were confirmed using independent PCR amplifications and sequenced in both directions
- DDR2 mutations within coding exons were screened from genomic DNA obtained from patient's tumor sample using Sanger sequencing method as described (6).
- the coding exons were designated based on mRNA transcript variant 2 from DDR2 gene (NM_ 006182). Mutations( based on SNP locations) were verified manually with comparison made to the matched normal sequence.
- FISH fluorescence in situ hybridization
- Chromatin was counterstained with DAPI (0.3 ⁇ g/mL in Vectashield; Vector Laboratories, Burlingame, CA). Analysis was performed using a fluorescence microscope with single interference filter sets for green (SpectrumGreen), orange (SpectrumOrange), and blue (DAPI) bandpass filters. Monochromatic images were captured and merged using a CytoVision workstation (Applied Imaging, San Jose, CA). Cells with single or split signals were defined as those with gene rearrangement. Those with both signals close or overlapping were negative for rearrangement.
- Total nucleic acids were extracted from five tissue sections using an SPRITE total nucleic acid extractor (Beckman Coulter). Each section was individually extracted and gDNA isolated. Briefly, sections were incubated in 200 of lysis buffer for 1 hour at 85 °C followed by 30 of proteinase K for 1 hour at 55°C, producing a total nucleic acid fraction.
- gDNA was isolated from the sections and cleaned for array comparative genomic hybridization (aCGH) and matrix-assisted laser desorption/ionization-time-of-flight mass spectroscopy single-nucleotide polymorphism analysis using a standard column cleanup with RNase treatment to remove RNA from the total nucleic acid fraction (DNeasy blood and tissue kit; QIAGEN) and adapted from an FFPE DNA preparation for oligonucleotide array- based CGH for a gDNA analysis protocol (Agilent Technologies). gDNA quantity and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific). The mean gDNA quantity recovered per tissue section was 10,340 ng. The mean sample purity (1.81) as assessed using a 260:280 wavelength ratio was within accepted DNA-purity levels (28). Individual gDNA preparations from each section were then pooled for use. Immunohistochemistry
- An automated stainer (Dako) was used for immunohistochemical staining of tumor sections. Five-micrometer sections of FFPE tumor were deparaffinized and hydrated, and antigen retrieval was done (pH 6.0, Dako). Cell pellets from NSCLC cell lines with high and low protein expression levels according to Western blotting relative to the levels in a panel of eight NSCLC cell lines were used as positive and negative controls for the staining. Peroxide blocking was performed using 3% methanol and hydrogen peroxide for 15 min.
- tissue slides were incubated with the primary antibodies phospho-PDGFRa (Y754, 1 :500), total PDGFRa (1 :500), c-Kit (1 :200), EphA2 (1 :500), phospho-EphA2 (Tyr594, 1 :500), phospho-c-Kit (Y719, 1 : 150), and p53 (1 :400) at room temperature for 1 hour.
- slides were incubated with Dako EnVision+ Dual Link reagent for 30 min at room temperature followed by a Dako chromogen substrate for 5 min and were then counterstained with hematoxylin for 5 min.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Urology & Nephrology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Hospice & Palliative Care (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Methods for determining whether a subject having cancer is a good candidate for a Src inhibitor therapy. For example, methods of the embodiments can comprise testing a sample from a subject to determine whether cancer cells from the subject have reduced BRAF activity and/or express a mutant BRAF protein. Methods for treating a subject having cancer with reduced BRAF kinase activity and/or that expresses a mutant BRAF protein by administering a Src inhibitor therapy are also provided.
Description
DESCRIPTION RESPONSE MARKERS FOR SRC INHIBITOR THERAPIES
[0001] The present application claims the priority benefit of United States provisional application number 61/621,747, filed April 9, 2012, the entire contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The invention was made with government support under Grant No. P50CA70907 awarded by the National Institutes of Health through the National Cancer Institute. The government has certain rights in the invention.
1. Field of the Invention
[0003] The present invention relates generally to the field of molecular biology and oncology. More particularly, it concerns methods for treating cancer and methods for predicting a response to anti-cancer therapies.
2. Description of Related Art
[0004] Lung cancer is the leading cause of cancer-related deaths worldwide. Researchers recently identified two molecular subpopulations of non-small cell lung cancer (NSCLC)— populations with epidermal growth factor receptor (EGFR) mutations and those with EML4-ALK translocations— for which treatment with targeted agents has produced profound clinical responses. This development has elicited a paradigm shift in treating lung cancer: the tumors' genetic characteristics dictate therapy. Nevertheless, definition of clinically relevant genetic aberrations is lacking in about 85% of NSCLC cases.
SUMMARY OF THE INVENTION
[0005] In a first embodiment there is provided a method for identifying a candidate for a Src inhibitor therapy comprising (a) testing a biological sample from a subject having a cancer to determine whether cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity relative to a wild type BRAF protein; and (b) identifying the subject as a good candidate for a Src inhibitor therapy if the sample from the subject indicates that cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity; or identifying the subject as a poor candidate for a Src inhibitor therapy if the sample
from the subject does not indicates that cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity.
[0006] In a further embodiment there is provided a method for identifying a good candidate for a Src inhibitor therapy comprising testing a biological sample from a subject having a cancer to determine whether cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity relative to a wild type BRAF protein, thereby identifying the subject as a good candidate for a Src inhibitor therapy.
[0007] In some aspects, testing the sample in accordance with the embodiments comprises measuring BRAF kinase activity in the sample. In certain aspects, testing a sample comprises determining whether cells of the cancer comprises a BRAF gene encoding a protein with reduced kinase activity. For example, the BRAF gene can encode a protein comprising an amino acid substitution or deletion at position Y472 (e.g., a Y472C amino acid substitution). Thus, in some aspects, testing a sample comprises determining all or part of a BRAF gene sequence in the sample. In some aspects, a sample is a blood sample, a tissue sample, a lymph sample or a saliva sample.
[0008] Certain aspects of the embodiments concern identifying a subject that is a good candidate for a Src inhibitor therapy, such as a subject that will have a favorable anticancer response to a Src inhibitor therapy. For example, a good candidate for a Src inhibitor therapy can be, without limitation, a subject that is predicted to have reduced in tumor size or burden, reduced tumor growth, reduced tumor-associated pain, reduced cancer associated pathology, reduced cancer associated symptoms, cancer non-progression, cancer remission, reduced cancer metastasis, or increased survival time in response to a Src inhibitor therapy. In further aspects, a method of the embodiments comprises providing a report, such as a written or electronic report, indicating whether the subject is a good candidate or a poor candidate for a Src inhibitor therapy. In some cases, such a report is transmitted to a third party such as a hospital, a doctor, an insurance company or a healthcare provider.
[0009] In a further embodiment, there is provided a method for treating a subject having a cancer, wherein it was determined that cancer cells from the subject comprise reduced BRAF kinase activity relative to a control level, the method comprising administering a Src inhibitor therapy (e.g., a Src kinase inhibitor) to the subject. For example, a method can comprise administering a Src inhibitor therapy 1, 2, 3, 4, 5, 6 or more
times to the subject over a period of days, weeks or months. In further aspects, a method of the embodiments further comprises administering at least a second anti-cancer therapy to the subject, such as chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy, anti- angiogenic therapy or cytokine therapy. Thus, in some aspects, a composition is provided for the treatment of a subject having cancer comprising cancer cells with reduced BRAF kinase activity relative to a control level, the composition comprising a therapeutically effective amount of a Src inhibitor.
[0010] In yet a further embodiment there is provided a method for treating a subject having a cancer comprising administering to the subject an effective amount of a Src inhibitor in conjunction with (e.g., before, after or concurrently with) a RAF inhibitor. For example, the Src inhibitor can be a Src kinase inhibitor, such as bosutinib, saracatinib, danusertib, VP-BHG712, quercetin, PCI-32765, KX2-391, AP23451 or dasatinib. In certain aspects, the RAF inhibitor is a BRAF inhibitor, such as a BRAF specific inhibitor. Examples of RAF inhibitors include, without limitation, vemurafenib, GSK2118436 and sorafenib. In further aspects, a composition is provided for the treatment of a subject having cancer comprising a therapeutically effective amount of a Src inhibitor and a RAF inhibitor.
[0011] In certain aspects a subject of the embodiments is a subject having or diagnosed with a cancer, such as a lung, breast, brain, prostate, spleen, pancreatic, cervical, ovarian, head and neck, esophageal, liver, skin, kidney, leukemia, bone, testicular, colon, or bladder cancer. For example, the cancer can be a lung cancer such as a non-small cell or a metastatic lung cancer.
[0012] In certain aspects, a Src inhibitor therapy of the embodiments is a Src kinase inhibitor. For example, the Src kinase inhibitor can be bosutinib, saracatinib, danusertib, VP-BHG712, quercetin, PCI-32765, KX2-391, AP23451 or dasatinib. [0013] In still a further embodiment there is provided computer-readable medium that, when executed by a computer, causes the computer to perform operations comprising (a) receiving information corresponding to a BRAF kinase activity in a sample from a subject having a cancer; and (b) determining a relative level of BRAF kinase activity compared to a reference level, wherein reduced BRAF kinase activity relative to the reference level indicates the subject is a good candidate for a Src inhibitor therapy. For example, the information corresponding to a BRAF kinase activity can comprises all or part of a BRAF
gene sequence. Thus, in some aspects, determining a relative level of BRAF kinase activity comprises determining a change in all or part of a BRAF gene sequence compared to a reference sequence, wherein a change in the BRAF gene sequence corresponding to reduced BRAF activity indicates that the subject is a good candidate for a Src inhibitor therapy. For example, a reference level of the embodiments can be all or part of a wild type BRAF gene sequence or all or part of a BRAF gene sequence corresponding to protein with increased or decreased kinase activity relative to a wild type sequence. In still further aspects, receiving information in accordance with the embodiments comprises receiving from a tangible data storage device information corresponding to a level of BRAF kinase activity in a sample from a subject having a cancer.
[0014] In further aspects a tangible computer-readable medium of the embodiments further comprises a computer-readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising: sending information corresponding to the relative level of BRAF kinase activity to a tangible data storage device. In yet further aspects, a tangible computer-readable medium comprises a computer-readable code that, when executed by a computer, causes the computer to perform operations further comprising (c) calculating a diagnostic score for the sample, wherein the diagnostic score is indicative of the probability that the subject will respond to a Src inhibitor therapy (e.g., whether the subject is a good candidate for a Src inhibitor therapy). [0015] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.
[0016] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.
[0017] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
[0018] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the
detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0020] FIG. 1. Y472CBRAF is biochemically similar to G466VBRAF but different from 600EBRAF. A) Kinase activity in Flag-tagged BRAF proteins with the noted mutations. After immunoprecipitation (IP) from COS7 cells and incubation with kinase-dead MEK (substrate) and ATP, MEK phosphorylation was measured using Western blotting and quantitated with ImageJ. (B-D) The effect of the expression of mutated BRAF proteins on MEK and ERK activity in intact cells. Western blotting was performed in (B) COS7, (C) H661, and (D) H226 cells transfected with BRAF constructs. E) The activity of CRAF in H661 cells transfected with mutated BRAF, as measured by incubating immunoprecipitated CRAF with kinase-dead MEK (substrate) and ATP. F) CRAF kinase activity and BRAF-CRAF binding, as measured in COS7 cells transfected with FLAG-tagged BRAF with the indicated mutations by kinase activity (IVKA) or IP of CRAF followed by blotting with anti-Flag and anti-BRAF antibodies (binding). WT, wild-type.
[0021] FIG. 2. NSCLC cells with an inactivating BRAF mutation undergo senescence when exposed to dasatinib. A-D) Biological effects of dasatinib in NSCLC cells with BRAF mutations. NSCLC cells were incubated with 150 nM dasatinib or a control vehicle for 72 hours followed by β-galactosidase (A) staining and (B) quantification, (C) cell cycle analysis using propidium iodide staining and FACS analysis, or (D) BrdU incorporation. E) The effect of dasatinib on downstream signaling as measured by Western blotting of NSCLC cells incubated with 150nM dasatinib for 24 hours. (F) The reversibility of dasatinib-induced senescence. Call2T cells were incubated with 150nM dasatinib or fresh medium (vehicle, but no drug) for the indicated times (left panel) and senescence was measured with β-galactosidase staining using light microscopy. *P <0.05. **P <0.001.
[0022] FIG. 3. Expression of kinase inactive and active BRAF leads to sensitivity and resistance to dasatinib, respectively, in NSCLC cells. (A, B, D) Cell viability was assayed following dasatinib treatment in NSCLC cells transfected with BRAF constructs. Following transfection with the noted BRAF constructs (A) H661, (B) H226, and (D) HI 666 cells were and incubated with dasatinib at increasing doses for 72 hours and cell viability was estimated using the MTT assay. The half-maximal inhibitory concentration (IC50) of dasatinib was calculated for H661. C) Apoptosis in H661 cells expressing BRAF with the noted mutations as measured using the terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling assay. [0023] FIG. 4. Dasatinib indirectly inhibits CRAF activity in cells with an inactivating BRAF mutation but not in cells with ^BRAF or 600EBRAF. A) Kinase activity of isolated BRAF and CRAF incubated with dasatinib. Purified recombinant BRAF and CRAF were incubated with 100 nM dasatinib in the presence of inactive MEK-1 and ATP. Kinase activity was measured by phosphorylation of MEK-1. B) The effect of dasatinib on the kinase activity of CRAF in intact cells expressing various BRAF constructs. COS-7 cells were transfected with Flag-tagged CRAF along with HA-tagged BRAF constructs with the noted mutations 24 hours prior to dasatinib treatment. Transfected cells were incubated with 150 nM dasatinib or vehicle for 24 hours followed by immunoprecitation with an anti-Flag antibody. The resulting lysate was subjected to a Western blot with anti-Flag and anti-HA antibodies (lower two rows) and CRAF kinase activity was measured by incubating Flag- immunoprecipitated CRAF with kinase-dead MEK-1 (substrate) and ATP (IVKA, top row). MEK activation was quantitated and normalized with total FLAG. (C) The effect of CRAF knock down (KD) on NSCLC cell number in cells with endogenous BRAF mutations. Untransfected Catl2T, H1666, H322, and H661 cells were incubated with CRAF siRNA, and their viability was estimated using an MTT assay. In all cases, Western blotting was used to confirm CRAF knockdown. At 120h Call2T cells lost dasatinib sensitivity and CRAF expression recovered.
[0024] FIG. 5. Dastinib induces RAF dimerization in NSCLC cells which is necessary for sensitivity in cells expressing kinase impaired BRAF. (A) Dasatinib induced RAF dimerization. NSCLC cells were incubated with 150 nM dasatinib for 72 hours then CRAF was immunoprecipitated. The resulting lysate was split with part used for blotting with antibodies to BRAF or CRAF (to measure dimerization) and part used to measure CRAF
kinase activity using MEK as a substrate. (B-C) Introduction of a mutation that interferes with RAF dimerization abrogates the effects of kinase deficient BRAF on dasatinib sensitivity. H661 cells transfected with the BRAF constructs as indicated were subjected to Western blotting (B) and an MTT assay (C). [0025] FIG. 6. Dasatinib enhances the cytotoxicity of PLX4032 in cancer cells resistant to dasatinib. Dasatinib-resistant NSCLC cells were incubated with single agent dasatinib (50-600 nM) or single agent PLX5032 (1000- 12000 nM) or in combination of both (dasatinib:PLX4032 = 1 :20) for 72 h, and their viability was estimated using an MTT assay.
[0026] FIG. 7. (A) Radiographic images of PX's primary lung tumor and paraspinal metastasis, demonstrating that only a residual lung nodule remains over 3 years after the completion of treatment with dasatinib alone for 12 weeks. (B) Kaplan-Meier overall survival and progression free survival curves for the 34 patients enrolled on the dasatinib phase 2 study at a median of 36 months of follow up.
[0027] FIG. 8. Alignment of the human BRAF (Homo; ; SEQ ID NO:3) amino acid sequence with that of mice (Mus; SEQ ID NO: L>, rats (Rattus; SEQ ID NO:2), chickens (Gallus; SEQ ID NO:4), and frogs (Xenopus; ; SEQ ID NO:5). The tyrosine residue marked in red represents the Y472 site in humans, which is conserved in all tested evolutionary distant organisms.
[0028] FIG. 9. NSCLC cells with kinase inactive BRAF mutations did not undergo apoptosis when exposed to 150nM dasatinib for 72 hours as measured by TUNEL staining.
[0029] FIG. 10. Cells with inactive BRAF undergo senescence in the presence of dasatinib. Immunofluorescence microscopy was done with Call2T, HI 666 (A), H661 and H2987 (B) cell lines treated with vehicle or 150 nM dasatinib for 72 hours. Cells were labeled with senescence-associated heterochromatin foci marker HPl-γ and counterstained with DAPI. Induction of HPl-γ in Dasatinib treated cells is significantly higher in Call2-T and HI 666. (C) Fold change in HPl-γ expressing cells in dasatinib treated group compared to vehicle control in three independent experiments.
[0030] FIG. 11. HI 666 cells were incubated with 100 nM dasatinib for the indicated times followed by replacement with fresh media (schedule per left panel) and senescence was quantitated with β-galactosidase staining using light microscopy. (*P value <0.05).
[0031] FIG. 12. H661 cells transfected with inactivating BRAF mutations show increased sensitivity to dasatinib. H661 cells were transfected with the noted BRAF constructs and incubated with increasing doses of dasatinib for 72 hours. Their viability was estimated using an MTT assay. These data were used to generate the data presented in FIG. 3A.
[0032] FIG. 13. Transfection with mutant or wt BRAF does not affect cell number. HI 666 and H661 cells were transfected with the noted BRAF constructs and cell number was estimated 72 hours later using the MTT assay.
[0033] FIG. 14. NSCLC cells with an inactivating BRAF mutation are sensitive to CRAF knockdown (KD). H661 cells expressing the noted BRAF proteins were incubated with CRAF siRNA, and their viability was estimated using an MTT assay. In all cases, Western blotting was used to confirm CRAF knockdown.
[0034] FIG. 15. Kinase inhibitor- induced RAF dimerization does not result in drug sensitivity or senescence. NSCLC cells were incubated with the noted drugs (dasatinib: 150 nM, nilotinib: 2 μΜ, bosutinib: 1.5 μΜ and AZD0530: 5 μΜ respectively) for 72 hours followed by immuoprecipitation with CRAF from Call2T cells (A), MTT assay (B), or β- galactosidase staining (C).
[0035] FIG. 16. Dasatinib does not have any BRAF mutation-specific changes in BAD or JNK phosphorylation. The noted NSCLC cell lines were incubated with 150 nM dasatinib for 72 hours followed by Western blotting with the noted antibodies.
[0036] FIG. 17. Inhibition of c-Src does not affect cell viability in NSCLC harboring a kinase inactive BRAF mutation. H1666 cells were incubated with AZD0530, c-Src siRNA, or controls. (A and B) Western blotting confirmed c-Src knock down or inhibition . (C) Neither c-Src siRNA nor controls affect cell viability 72 hours following transfection. (note: The MTT assay for AZD0530 is included in FIG. 15B).
[0037] FIG. 18. Dasatinib enhances the cytotoxicity of sorafenib in cancer cells resistant to dasatinib. Dasatinib-resistant head and neck cancer (top four panels) and NSCLC (bottom four panels) cells were incubated with 100 nM dasatinib and 1-10 μΜ sorafenib for 72 hours, and their viability was estimated using an MTT assay.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0038] Metastatic NSCLC is a common and fatal disease with a 4-year survival rate of only 2%, but personalized therapies targeting specific genetic aberrations in NSCLC tumors are remarkably successful. A patient was identified with stage IV NSCLC with long term disease control by single-agent dasatinib therapy alone. His tumor harbored a kinase inactivating Y472CBRAF mutation that was likely responsible for its unusual sensitivity to dasatinib. Although effective treatments are available for melanoma patients with activating BRAF mutations, no such therapies are available for cancer patients who harbor inactivating mutations. [0039] No inactivating BRAF mutations were identified in any other patients in the clinical trial, all of whom were nonresponders. As with previously characterized kinase inactivating BRAF mutations, Y472CBRAF expression led to CRAF, MEK, and ERK activation. Also, NSCLC cell lines with endogenous inactivating BRAF mutation underwent senescence when exposed to dasatinib; the cell lines' dasatinib sensitivity reversed with the overexpression of active BRAF. Whereas NSCLC cells transfected with an activating BRAF mutation were more resistant to dasatinib than were controls, transfection of NSCLC cells with kinase impaired BRAF led to their increased dasatinib sensitivity. These data and PX's pattern of clinical response are consistent with the conclusion that his tumor underwent senescence when exposed to dasatinib owing to its inactivating BRAF mutation. The mechanism of dasatinib-induced senescence and apoptosis in NSCLC cells expressing kinase impaired BRAF is unknown but may relate to increased RAF dimerization leading to ERK activation consistent with the paradigm of oncogene-induced senescence that occurs following moderate BRAF or KRAS activation (1 1). However, the modest effects on ERK suggest that ERK-independent pathways are involved as well. [0040] BRAF mutations occur in only about 4% of NSCLC cases but are more common in other tumors, such as melanoma (50%) and papillary thyroid cancer (40%) (19,20). The majority of BRAF mutations cause activation of the kinase (20). In NSCLC, 57% of mutations are remaining 43% are a mixture of kinase inactivating, activating, and uncharacterized mutations (19). CRAF mutations are rare in all cancers (21). [0041] Interactions between CRAF and BRAF are complex and incompletely understood despite several recent elegant studies in melanoma (4, 5, 22, 23). The pathway is
not linear and is further complicated by the multiplicity of signaling molecule components; BRAF and CRAF are similar to each other but do not function identically and are not interchangeable and feedback pathways lead to inactivation of upstream components of the pathway (i.e., a negative feedback amplifier) (24). Additionally, MEK is not the only RAF substrate (25).
[0042] The complexity of BRAF-CRAF interactions is epitomized by the RAF inhibitor paradox. Specifically, inhibition of BRAF activity in cells that express 600EBRAF results in expected inhibition of ERK activity and subsequent apoptosis; melanomas with 600EBRAF respond clinically to BRAF inhibition (26). Paradoxically, BRAF inhibition in cells with active RAS leads to ERK activation via activation of CRAF (4). These observations are supported by previous research demonstrating that CRAF and BRAF heterodimers are more active than is either the CRAF or BRAF homodimer even when the BRAF protomer has an inactivating mutation (23).
[0043] These complex interactions of RAS, BRAF, and CRAF explain the existence and function of inactivating BRAF mutations in cancer cells, which superficially seem to be contrary to natural selection. Activation of CRAF not only allows cells expressing kinase impaired BRAF to survive but also may further promote the cancer phenotype by activating non-ERK-dependent pathways or promoting aneuploidy (25, 27). CRAF activation may also be responsible for BRAF-inhibitor-induced squamous cell carcinomas (26). [0044] Although much of the research involving BRAF has focused on melanoma, the BRAF pathway undoubtedly is active and important in NSCLC. RAS is commonly activated in NSCLC via upstream growth factor receptors or activating mutations. Active KRAS signals predominantly through CRAF, not BRAF (28). The role of CRAF in NSCLC has yet to be elucidated, although CRAF is overexpressed in NSCLC cells, and its forced overexpression leads to lung adenomas in a transgenic mouse model (29).
[0045] Oncogene-induced senescence occurs after activation of oncogenes such as RAS and RAF. Classically, it is mediated by activation of the ρΙό^^/ΕΗ) and/or pi 4ARF/p53 tumor suppressor pathway. Senescence also can be induced and mediated by other pathways, such as those involving c-Src, STAT3, c-Myc, FOX04, Chk2, and c-Jun-N-terminal-kinase. In the present study, dasatinib induced senescence in HI 666 and Call2T cells but induced apoptosis in H661 cells into which were transfected kinase impaired BRAF mutations.
Possible reasons for this discrepancy are that the absolute level of endogenous w BRAF, the level of mutant BRAF, or the w mutant BRAF ratio may influence the outcome of dasatinib- based treatment. The level of mutant BRAF expression was higher in the transfected cells, which also harbored a full complement of endogenous "'BRAF, than in non-trans fected cells. [0046] Occasionally, spontaneous tumor regression occurs in melanoma and renal cell carcinoma cases and is thought to be immune mediated. Also, the activating V600EBRAF mutation in melanoma may induce an immune response. Although such a possibility cannot be excluded in the case of PX, spontaneous tumor regression is very rare in NSCLC cases. Instead, the dasatinib sensitivity of NSCLC cell lines with kinase impaired BRAF mutations is consistent with the patient having experienced a direct antitumor effect of dasatinib rather than an immune-mediated mechanism.
[0047] Other potential mechanisms not fully explored in these studies are the roles of other dasatinib targets in mediating dasatinib sensitivity. Although no protein expression has been linked to dasatinib sensitivity, Sos et al. demonstrated that copy-number gains influence sensitivity of NSCLC cells to kinase inhibitors (7). H322 cells, which have c-Src amplification, are sensitive to dasatinib. PX's tumor had copy -number gains in several dasatinib targets. Because live tumor tissue specimens are not available from PX, determining whether his tumor was dependent upon HCK, DDR1, EPHA3, or ARG for survival is impossible (30). [0048] In conclusion, it was demonstrated that the kinase inactive mutation
Y472CBRAF functions similarly to other known inactivating BRAF mutations and was likely responsible for PX's response to dasatinib. Moreover, the potential for synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options. Given that dasatinib and BRAF inhibitors are in clinical use, this work has potential for direct clinical applications.
I. Examples
[0049] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the
present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1 - Patient's Tumor Analysis [0050] In a Phase 2 study of dasatinib in 34 patients with systemic therapy-naive stage IV NSCLC the sole responder was a male former smoker (PX) who had a profound, durable response (2). Over the 12 weeks of dasatinib-based therapy, PX had a partial response as assessed by both tumor size and metabolic activity and his metastatic tumor (in paraspinal muscle) continued to shrink after therapy was stopped. At the end of therapy, the diameter of the metastasis was 2.8 cm, with a standardized uptake value (SUV) of 17. At 17 weeks, accurately measuring the metastasis on a computed tomography (CT) scan was difficult, but the SUV was 1 1. At 21 weeks, the SUV was 4.5. At 32 weeks, the mass was undetectable on CT and positron emission tomography scans (2). Subsequent follow up shows that PX remains free of active cancer 4 years after the initial diagnosis and has not received any other cancer therapy. PX still has a 2-cm lung nodule that has no detectable metabolic activity on PET and that has been stable on CT scans for 4 years (FIG. 7A). The median progression free survival was 1.4 months and the median overall survival was 15.6 months (FIG. 7B). Additional studies of PX's tumor tissue were performed to identify the underlying mechanism of dasatinib sensitivity. [0051] PX's tumor did not harbor any EGFR or KRAS mutations by intron-based polymerase chain reaction (PCR) οΐΚ-Ras exons 1 and 2 (codons 12, 13, and 61) and EGFR exons 18-21 as previously published (2). Likewise, no ALK gene rearrangements were detected by fluorescence in situ hybridization; no c-Src mutations were detected by intron- based PCR of exons 7-10; nor were any discoidin domain receptor 2 (DDR2) mutations detected using conventional Sanger Sequencing of all exons as previously described (6). Immunohistochemistry revealed that the tumor did express total and phosphorylated EphA2, c-Kit, and PDGFRa (Table 1). PX did not harbor any germ line BRAF or KRAS mutations by intron-based PCR of BRAF exons 1 1 and 15 and K-Ras exons 1 and 2 (codons 12, 13, and 61) of DNA isolated from his peripheral blood lymphocytes. [0052] To identify novel mutations or changes in gene copy number in PX's tumor, the MassARRAY system (Sequenom) was used and aCGH performed. No mutations were
detected among the 40 genes tested (Table 2). Using aCGH, several regions of increased and decreased copy numbers were detected (Table 3). Increased copy numbers of the known direct dasatinib targets HCK, DDR1, EPHA3, and ARG (ABL2) were also detected. No copy number changes for LYN, FGR, FYN, SRC, DDR2, EPHB1, EPHB2, EPHB3, EPHA1, EPHA2, EPHA4, TNK2, PTK6, GAK, KIT, PDGFR, KRAS, EGFR, or BRAF were detected.
[0053] Table 1: Immunchistochemistry scores for PX's tumor.
[0054] Table 2: Genes analyzed using mass spectroscopy single-nucleotide polymorphism analysis.
Amino acid Nucleic acid change
position tested
Amino acid Nucleic acid change position tested
Amino acid Nucleic acid change position tested
Amino acid Nucleic acid change position tested
Amino acid Nucleic acid change position tested
Amino acid Nucleic acid change position tested
[0055] Table 3: Copy number variation for genes associated with dasatinib targeting and/or NSCLC.
* Dasatinib target-associated gene.
[0056] Table 4: Mutational statuses of patients with NSCLC treated with dasatinib.
RECIST, Response Evaluation Criteria in Solid Tumors; AC, adenocarcinoma; PD, progressive disease; NE, not evalauted; SD, stable disease; PR, partial response; SCC, squamous cell carcinoma.
Example 2 - Identification of a Novel Inactivating Mutation in BRAF
[0057] Because the Sequenom MassARRAY technology is limited in that can only identify candidate mutations in which assays are specifically designed and given the known role of BRAF in oncogene-induced senescence, exons 1 1 and 15 of the BRAF gene were sequenced. These two exons possess many known mutations not included in the panel. The identified mutation Y472CBRAF, has not been described previously and occurs in a highly conserved region of exon 1 1 (FIG. 8). The BRAF gene was also analyzed in 19 patients from the original clinical trial for whom DNA adequate for analysis was available and no other inactivating mutations were found (Table 4). [0058] To determine the functional significance of Y472CBRAF, site-directed mutagenesis was used to create Y472CBRAF, G466 BRAF (kinase-impaired), and 600EBRAF (constitutively active) in a Flag-tagged ^BRAF construct. The constructs were transfected into COS7 cells, the Flag-tagged proteins isolated and tested for kinase activity. As expected, V600EBRAF had increased kinase activity and G466VBRAF had reduced kinase activity. Y472CBRAF showed severely reduced kinase activity that was less than 10% that of ^BRAF (FIG. 1A).
Example 3 - Lung Cancer Cells Expressing Y472CBRAF Express Activated MEK, ERK, and CRAF
[0059] Cancer cells that express kinase impaired BRAF mutations still activate MEK and ERK via trans activation of CRAF (4, 5). To determine whether Y472CBRAF functions similarly, COS7, H226, and H661 cells (all with ^BRAF) were transfected with the BRAF construct panel and their ERK and MEK activity measured (FIG. IB-ID). As expected, transfection with 600EBRAF resulted in phosphorylation of ERK and MEK to levels markedly above those in cells transfected with wtBRAF. Y472CBRAF and G466VBRAF both activated MEK and ERK to levels at or above those observed after transfection with ^BRAF. Cells transfected with ^BRAF had similar levels of total BRAF compared to cells transfected with mutant BRAF (FIG. 1C and ID).
[0060] To determine whether Y472CBRAF transactivates CRAF, CRAF kinase activity was assessed in H661 cells transfected with the BRAF constructs panel. Cells expressing
G466VBRAF or Y472CBRAF CRAp actiyity man djd mQs e ceUs expressing ^BRAF or (FIG. i ImE). τ L ;ik1 ewise, c _eilils express ;ing G466V Bn>nRATF? o„„r Y Y447/2CL TBRAF had higher
BRAF-CRAF binding than did cells expressing BRAF or VWJU¾RAF (FIG. IF). These results confirmed that Y472CBRAF functions in the RAS/RAF/MEK/ERK pathway similarly to previously characterized BRAF proteins with inactivating mutations.
Example 4 - Dasatinib Leads to Senescence and Apoptosis in Lung Cancer Cells
Expressing Kinase impaired BRAF
[0061] To determine whether Y472CBRAF was related to PX's response to dasatinib, w a panel of NSCLC cell lines was tested for their sensitivity to dasatinib. After extensive searching in multiple databases, only two NSCLC cell lines were identified with known inactivating BRAF mutations: H1666 and Call2T. These two lines, along with one cell line expressing ^BRAF (H322), were sensitive to dasatinib (Table 5). Researchers previously characterized H322 cells as having c-Src amplification, which drives their sensitivity to dasatinib (7). All other lines tested were resistant to dasatinib, including those with activating BRAF mutations (Table 5).
[0062] Significant apoptosis was not observed when H1666 or Call2T cells were treated with dasatinib (FIG. 9), but the cells did undergo cell-cycle arrest with an increased proportion of cells in Gl, decreased proliferation with reduced BrdU incorporation, and they stained for β-galactosidase and HPI-gamma, indicating senescence (FIGs. 2A-2D and 10). Consistent with this finding, dasatinib reduced phosphorylated Rb in HI 666 and Call2T (FIG. 2E). [0063] To determine whether dasatinib-induced senescence was reversible, Call2T and HI 666 cells were incubated with dasatinib for 6-96 h followed by drug removal and measurement of β-galactosidase expression (FIGs. 2F, 1 1). Induction of significant tumor cell senescence required 72 h of exposure to dasatinib and was largely irreversible by 96h.
[0064] To further study the effects of inactivating BRAF mutations on dasatinib sensitivity, H661 and H226 cells were transfected with the panel of BRAF constructs. Expression of the inactivating mutations increased the cells' sensitivity to dasatinib, whereas expression of 600EBRAF induced further resistance (FIGs. 3A, 3B, and 12). In contrast HI 666 and Call2T cells, which have endogenous BRAF inactivation (G466 BRAF), H661 cells, transfected with kinase impaired BRAF, underwent apoptosis and not senescence when exposed to dasatinib (FIG. 3C).
[0065] To confirm the importance of the inactivating BRAF mutations in mediating dasatinib sensitivity in HI 666 cells, these cells were transfected with kinase active BRAF (i.e., 600EBRAF or ^BRAF) and treated them with dasatinib (FIG. 3D). Overexpression of kinase active BRAF increased resistance to dasatinib, confirming the role of kinase inactive BRAF in mediating dasatinib sensitivity. None of the constructs in the panel of BRAF constructs had a significant effect on cell number in untreated cells (FIG. 13).
[0066] Table 5: Lung cancer cell sensitivity to dasatinib
BRAF Kinase
mutation Dasatinib activity
Cell line BRAF type IC50 (nM) (fold wt)* Other known mutations or polymorphisms
H1666 G466V Inactivating 158 0-65 CDKN2A, STKl 1
H322 wt wt 142 1 TP53, CDKN2A, STKl 1
Cal-12T G466V Inactivating 238 0-65 TP53, CDKN2A
H661 wt wt 1484 1 TP53, CDKN2A, SMARCA4
H460 wt wt >2500 1 KRAS, CDKN2A, STKl 1, PIK3CA
H1568 wt wt 3321 1 None known
H226 wt wt >5000 1 TP53
A549 wt wt >5000 1 KRAS, CDKN2A, SMARCA4, STKl 1
H522 wt wt >5000 1 TP53
H2087 L597V Activating >5000 64 NRAS, TP53, DGKB, DGKB, PTPRT, ATM, BAI3, HECWl,
HEPH, NLRP4, NTRK3, PDZRN4, PTPRM
H1755 G469A Activating >5000 266 KRAS, TP53
H1395 G469A Activating >5000 266 STKl 1, NLRPl, RTPRT, ATM, BIRC6, NLRP4, TRIM36
HCC364 V600E Activating >5000 478 None known
H2405 In- frame Unknown >8000 ND TP53
deletion
*Based on published values determined using an in vitro kinase assay (33, 34). ND, not done.
Example 5 - Dasatinib Indirectly Inhibits CRAF
[0067] The mechanism by which dasatinib induces senescence in NSCLC cells with kinase-deficient BRAF is unknown. It was hypothesized that dasatinib induces senescence by affecting CRAF function (8). Although it was found that dasatinib did not directly affect CRAF or BRAF kinase activity at relevant concentrations (FIG. 4A), which is consistent with published studies (7, 9), it led to decreased CRAF activity in intact cells that express kinase inactive BRAF (FIG. 4B). To establish the importance of CRAF in NSCLC cells with kinase impaired BRAF, CRAF expression was knocked-down using siRNA in NSCLC cells with kinase active or inactive BRAF. CRAF knockdown in H1666 (G466VBRAF) and Call2T (GA66 BRAF), but not H322 (wtBRAF) or H661 (wtBRAF) cells affected their viability as estimated using an MTT assay (FIG. 4C), although the effects on Call2T cells were modest. Given the limited number of cells lines expressing endogenous mutated BRAF, a less physiologic approach was used and H661 cells were transfected with kinase active or inactive BRAF. Only cells expressing kinase-deficient BRAF showed reduced cell number after treatment with CRAF siRNA (FIG. 6).
Example 6 - Dasatinib Induces RAF Dimerization
[0068] Consistent with the recent findings of Packer et. al. it was found that dasatinib induced RAF dimerization in cells with a KRAS mutation (A549) (10). In addition, dasatinib induced RAF dimerization in NSCLC cells with kinase impaired BRAF (FIG. 5A). Although dasatinib caused a modest inhibition of RAF kinase activity, when corrected for the increase in total RAF dimers, there was no net inhibition of RAF in Call2T and H1666 cells. Similarly, ERK was activated and p21 expression induced after dasatinib treatment in cells with kinase impaired BRAF consistent with oncogene induced senescence that is observed with KRAS or BRAF activation (FIG. 2E) (1 1). ERK and MEK were inhibited in H661 and A549 cells, which is consistent with their lack of senescence following dasatinib exposure.
[0069] To further investigate the role of BRAF/CRAF heterodimerization in dasatinib-induced senescence, a BRAF mutant that prevents dimerization (R509H5R^F) was transfected into NSCLC cells with endogenous wtBRAF either as a single mutation or in cis with Y472CBRAF (FIG. 5B) (12). As before, the expression of Y472CBRAF led to the activation of ERK and increased sensitivity to dasatinib. The addition of R509HBRAF to Y472CBRAF
inhibited BRAF's ability to activate ERK. NSCLC cells expressing the double mutations were not more sensitive to dasatinib than those expressing ^BRAF (FIG. 5C).
[0070] Drug-induced RAF dimerization alone was not adequate to induce senescence in Call2T or H1666 cells. Nilotinib induced more robust BRAF/CRAF dimerization than did dasatinib in Call2T cells but did not induce senescence (FIG. 15). Together these experiments demonstrate that dasatinib-induced RAF dimerization is essential, but not sufficient for dasatinib sensitivity.
[0071] A preliminary investigation of ERK-independent pathways downstream of RAF demonstrated no mutation-specific changes in BAD or J K (13). Activated Aurora A and PLK1 were not detected by Western blotting (FIG. 16) (14). Src family kinase inhibition with AZD0530 or knockdown with siRNA was not adequate to induce significant senescence or cytotoxicity (FIGs. 15 and 16).
Example 7 - The Combination of a BRAF Inhibitor and Dasatinib Leads to Synergistic
Cytotoxicity in Cancer Cells Resistant to Dasatinib [0072] The sensitivity of cancer cells with inactivating BRAF mutations to dasatinib suggests BRAF's synthetic lethality with a dasatinib target. Cell lines were treated with ^BRAF and marked resistance to dasatinib (15,16) with dasatinib plus the pan-RAF inhibitor sorafenib or dasatinib plus the BRAF inhibitor PLX4032 (vemurafenib) and then measured the treatment's effect on these lines' viability in vitro. In all cases, dasatinib enhanced the effect of the RAF inhibitors at clinically relevant doses (FIGs. 6 and 18) and formal analysis demonstrated synergy. Although specific CRAF and pan-RAF inhibitors may be as effective as dasatinib in treating cancers with inactivating BRAF mutations, no credible direct CRAF inhibitors are currently in clinical development, and pan-RAF inhibitors have poor in vivo activity and are not specific (17, 18). Example 8 - Discussion
[0073] Metastatic NSCLC is a common and fatal disease with a 4-year survival rate of only 2%, but personalized therapies targeting specific genetic aberrations in NSCLC tumors are remarkably successful. A patient was identified with stage IV NSCLC with long term disease control by single-agent dasatinib therapy alone. His tumor harbored a kinase inactivating Y472CBRAF mutation that was likely responsible for its unusual sensitivity to
dasatinib. Although effective treatments are available for melanoma patients with activating BRAF mutations, no such therapies are available for cancer patients who harbor inactivating mutations.
[0074] No inactivating BRAF mutations were identified in any other patients in the clinical trial, all of whom were nonresponders. As with previously characterized kinase inactivating BRAF mutations, Y412CBRAF expression led to CRAF, MEK, and ERK activation. Also, NSCLC cell lines with endogenous inactivating BRAF mutation underwent senescence when exposed to dasatinib; the cell lines' dasatinib sensitivity reversed with the overexpression of active BRAF. Whereas NSCLC cells transfected with an activating BRAF mutation were more resistant to dasatinib than were controls, transfection of NSCLC cells with kinase impaired BRAF led to their increased dasatinib sensitivity. These data and PX's pattern of clinical response are consistent with the conclusion that his tumor underwent senescence when exposed to dasatinib owing to its inactivating BRAF mutation. The mechanism of dasatinib-induced senescence and apoptosis in NSCLC cells expressing kinase impaired BRAF is unknown but may relate to increased RAF dimerization leading to ERK activation consistent with the paradigm of oncogene-induced senescence that occurs following moderate BRAF or KRAS activation (1 1). However, the modest effects on ERK suggest that ERK-independent pathways are involved as well.
[0075] BRAF mutations occur in only about 4% of NSCLC cases but are more common in other tumors, such as melanoma (50%) and papillary thyroid cancer (40%) (19,20). The majority of BRAF mutations cause activation of the kinase (20). In NSCLC, 57% of mutations are remaining 43% are a mixture of kinase inactivating, activating, and uncharacterized mutations (19). CRAF mutations are rare in all cancers (21).
[0076] Interactions between CRAF and BRAF are complex and incompletely understood despite several recent elegant studies in melanoma (4, 5, 22, 23). The pathway is not linear and is further complicated by the multiplicity of signaling molecule components; BRAF and CRAF are similar to each other but do not function identically and are not interchangeable and feedback pathways lead to inactivation of upstream components of the pathway (i.e., a negative feedback amplifier) (24). Additionally, MEK is not the only RAF substrate (25).
[0077] The complexity of BRAF-CRAF interactions is epitomized by the RAF inhibitor paradox. Specifically, inhibition of BRAF activity in cells that express 600EBRAF results in expected inhibition of ERK activity and subsequent apoptosis; melanomas with 600EBRAF respond clinically to BRAF inhibition (26). Paradoxically, BRAF inhibition in cells with active RAS leads to ERK activation via activation of CRAF (4). These observations are supported by previous research demonstrating that CRAF and BRAF heterodimers are more active than is either the CRAF or BRAF homodimer even when the BRAF protomer has an inactivating mutation (23).
[0078] These complex interactions of RAS, BRAF, and CRAF explain the existence and function of inactivating BRAF mutations in cancer cells, which superficially seem to be contrary to natural selection. Activation of CRAF not only allows cells expressing kinase impaired BRAF to survive but also may further promote the cancer phenotype by activating non-ERK-dependent pathways or promoting aneuploidy (25, 27). CRAF activation may also be responsible for BRAF-inhibitor-induced squamous cell carcinomas (26). [0079] Although much of the research involving BRAF has focused on melanoma, the BRAF pathway undoubtedly is active and important in NSCLC. RAS is commonly activated in NSCLC via upstream growth factor receptors or activating mutations. Active KRAS signals predominantly through CRAF, not BRAF (28). The role of CRAF in NSCLC has yet to be elucidated, although CRAF is overexpressed in NSCLC cells, and its forced overexpression leads to lung adenomas in a transgenic mouse model (29).
[0080] Oncogene-induced senescence occurs after activation of oncogenes such as RAS and RAF. Classically, it is mediated by activation of the p^^/Rb and/or pi 4ARF/p53 tumor suppressor pathway. Senescence also can be induced and mediated by other pathways, such as those involving c-Src, STAT3, c-Myc, FOX04, Chk2, and c-Jun-N-terminal-kinase. In the present study, dasatinib induced senescence in HI 666 and Call2T cells but induced apoptosis in H661 cells into which were transfected kinase impaired BRAF mutations. Possible reasons for this discrepancy are that the absolute level of endogenous wtBRAF, the level of mutant BRAF, or the w mutant BRAF ratio may influence the outcome of dasatinib- based treatment. The level of mutant BRAF expression was higher in the transfected cells, which also harbored a full complement of endogenous "'BRAF, than in non-trans fected cells.
[0081] Occasionally, spontaneous tumor regression occurs in melanoma and renal cell carcinoma cases and is thought to be immune mediated. Also, the activating V600EBRAF mutation in melanoma may induce an immune response. Although such a possibility cannot be excluded in the case of PX, spontaneous tumor regression is very rare in NSCLC cases. Instead, the dasatinib sensitivity of NSCLC cell lines with kinase impaired BRAF mutations is consistent with the patient having experienced a direct antitumor effect of dasatinib rather than an immune-mediated mechanism.
[0082] Other potential mechanisms not fully explored in these studies are the roles of other dasatinib targets in mediating dasatinib sensitivity. Although no protein expression has been linked to dasatinib sensitivity, Sos et al. demonstrated that copy-number gains influence sensitivity of NSCLC cells to kinase inhibitors (7). H322 cells, which have c-Src amplification, are sensitive to dasatinib. PX's tumor had copy -number gains in several dasatinib targets. Because live tumor tissue specimens are not available from PX, determining whether his tumor was dependent upon HCK, DDR1, EPHA3, or ARG for survival is impossible (30).
[0083] In conclusion, it was demonstrated that the kinase inactive mutation Y472CBRAF functions similarly to other known inactivating BRAF mutations and was likely responsible for PX's response to dasatinib. Moreover, the potential for synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options. Given that dasatinib and BRAF inhibitors are in clinical use, this work has potential for direct clinical applications.
Example 9 - Materials and Methods
Antibodies
[0084] Antibodies used in this study were phosphorylated SFK (pSFK, Y416), pERK 1/2 (T202/Y204), p21cip, Bcl2, total ERK, pMEK-1/2 (S217/221), phospho-EphA2 (Tyr594), and p-c-Kit (Y719) (Cell Signaling Technology); total CRAF (BD Biosciences); total BRAF, phospho-platelet-derived growth factor (pPDGFRa; Y754), total PDGFRa, c-Kit, Flag M2, and agarose-conjugated CRAF (Santa Cruz Biotechnology); Flag and β-actin (Sigma Chemical Co); and p53 (Dako). HPl-γ (Millipore); Anti mouse Alexa fluor 594 (Molecular Probes). Dasatinib, PLX4032 and sorafenib were purchased from Selleck Chemicals and prepared as 10 mM stock solutions in dimethyl sulfoxide.
Cytotoxicity Assay
[0085] Cytotoxicity in NSCLC cells was assessed using a 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described (31). Under each experimental condition, at least four independent wells were treated. Median effects of drugs on viability were calculated using the Chou-Talalay equation (32) with the CalcuSyn software program (Biosoft).
Senescence-Associated β-Galactosidase Staining
[0086] NSCLC cells were processed using a senescence-associated β-galactosdiase staining kit (Cell Signaling Technology) according to the manufacturer's instructions and visualized under an Olympus 1X71 phase microscope (Olympus America). In brief, upon completion of dasatinib-based treatment, cells were washed with phosphate-buffered saline to remove residual media and fixed. A β-galactosidase staining solution containing X-gal was then added to the fixed cells and incubated at 37°C overnight in a dry incubator without CO2. Fields with at least 100 cells were counted, in triplicate. BRAF and CRAF Kinase Assays
[0087] The kinase activity of immunoprecipitated endogenous BRAF and CRAF protein from NSCLC cells; purified recombinant proteins; or immunoprecipitated Flag- tagged BRAF or Flag-tagged CRAF protein expressed in COS-7 cells was measured using an in vitro kinase assay (IVKA) kit for RAF (Millipore). Purified recombinant BRAF and CRAF (Sigma Chemical Co.) and immunoprecipitated proteins (technique described below using anti-Flag, -HA, -BRAF, or -CRAF antibodies) were incubated with 100-250 μΜ ATP/Mg2+ along with 1 μg of inactive recombinant GST-MEK-1 at 30°C for 30 min and subsequently boiled with lx sample buffer to stop the reaction. MEK-1 activation was quantified by measuring the phospho-specific MEK-1 band (ImageJ software program; National Institutes of Health) after Western blotting (described below).
Cell-Cycle, Proliferation and Apoptosis Assays
[0088] For cell-cycle analysis, cells were harvested, fixed, and stained with propidium iodide, and their DNA content was analyzed using a cytofluorimeter and fluorescence-activated cell sorter (FACS) (FACScan; Becton Dickinson) and using the ModFit software program (Verity Software House) (31). To measure apoptosis, fixed cells
were subjected to terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TU EL) staining according to the manufacturer's procedure (APO-BRDU kit; Phoenix Flow Systems) and quantitated using the FACS (31). BrdU incorporation was measured according to the manufacturer's instruction (BrdU Flow Kits, BD Biosciences). Briefly, subconfluent cell cultures were treated with DMSO or 150 nM dasatinib for 72 hours, then labeled with 10 μΜ BrdU for 4 hr. Cells were trypsinized, fixed, and stained with FITC- conjugated anti-BrdU antibody and 7-Aminoactinomycin D (7-AAD). Samples were analyzed by two-dimensional flow cytometry to detect both fluorescein and 7-AAD.
Transfection of NSCLC Cells with a Small Interfering RNA [0089] Cells were harvested, washed, and suspended (106 cells/ 100 μΐ,) in
Nucleofector Solution V (Amaxa), and small interfering RNA (siRNA; 200 pmol/100 μί) was added to the cells. Cells were then electroporated using the Nucleofector program U24 (Amaxa) and diluted with a prewarmed 500-μΕ RPMI medium supplemented with 10% serum and plated onto 60-mm plates and the medium was changed 16 hours later. The c-Src and CRAF siRNAs were predesigned as sets of four independent sequences (siGENOME SMARTpool; Dharmacon). Controls were cells transfected with a nontargeting (scrambled) siRNA and mock-transfected cells (i.e., no siRNA).
Site-Directed Mutagenesis
[0090] Flag-tagged ^BRAF and 600EBRAF plasmids were provided by Dr. Walter Kolch (Systems Biology Ireland and The Conway Institute, University College Dublin). The ^BRAF construct was used as a template to create the Y472CBRAF , G466 BRAF, R509HBRAF and ™ ™∞BRAF mutations using the QuikChange XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The sense mutagenic primers used were 5'-GATCATTTGGAACAGTCTGCAAGGGAAAGTGGCATGGT -3' (SEQ ID NO: 6) for Y412CBRAF and 5'-GTGGGACAAAGAATTGGATCTGTATCATTTGGAACA GTC -3' (SEQ ID NO: 7) for G466W BRAF, 5'-
GTAGGAGTACTCAGGAAAACACACCATGTGAATATCCTACTCT-3 ' (SEQ ID NO:8) for R509HBRAF and Υ472™509¾/^ To confirm successful introduction of the mutations, six different plasmids for each mutant were Sanger sequenced.
Immunoprecipitation and Western Blot Analysis
[0091] For both Western blot and immunoprecipitation (IP) analysis, cells were lysed on ice, and the lysates were centrifuged at 20,000g for 5 min at 4°C as described previously (31). For IP, equal amounts of the protein cell lysate supernatant (500 mg) were precleared with protein A and G sepharose beads (Invitrogen). The precleared lysate was incubated with an IP antibody (anti-Flag or anti-CRAF) overnight. The immunoprecipitates were washed four times with an immunocomplex wash buffer (50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 1% Triton X-100, 1 mmol/L egtazic acid, 1 mmol/L ethylenediaminetetraacetic acid, 1% glycerol, 20 μg/mL leupeptin, 10 μg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium vanadate) and boiled with l x sample buffer for 5 min. For both the IP and Western blot analysis, equal protein aliquots were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, immunoblotted with a primary antibody, and detected using a horseradish peroxidase- conjugated secondary antibody (Bio-Rad Laboratories) and ECL reagent (Amersham Biosciences).
Clinical Tissue Specimens
[0092] Patients' tissue specimens were obtained in an MD Anderson Institutional Review Board-approved clinical trial in which all participants gave permission for testing of residual tumor tissue. For PX, residual tissue from a metastatic axillary lymph node resected 2 months before treatment was used. The metastasis was formalin-fixed and paraffin- embedded (FFPE) according to a standard protocol. One 4-μιη tissue section was cut and stained with hematoxylin and eosin. Histopathological tumor examination and visual estimation of the tumor area and tumor-cell percentage were conducted by a pathologist. The tissue section had a 2.5 x 1.8 cm lymph node extensively infiltrated with malignant epithelial cells representing -60 % of the whole tissue section. Five 10-μιη unstained tissue sections were cut and placed in sterile tubes for genomic DNA (gDNA) extraction and isolation.
Cell Culture
[0093] Call2T cells were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. All other human NSCLC cell lines were obtained from Drs. John Heymach (The University of Texas MD Anderson Cancer Center) and John Minna (The University of Texas Southwestern Medical Center) and maintained using standard cell culture
techniques (30). COS7 monkey kidney cells were obtained from Dr. Ho-Young Lee (MD Anderson). All cell lines were validated by cross-comparing their allelic short tandem repeat patterns generated using the PowerPlex 1.2 system (Promegl) with the American Type Culture Collection repository database. BRAF mutations were confirmed using Sanger sequencing of exons 1 1 and 15.
Immunofluorescence microscopy
[0094] Cells were fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.5% NP40 for 10 min at room temperature. Cover slips were blocked in 10% normal goat serum in PBS for 30 min, and incubated with the anti-ΗΡΙγ antibody (1 :200 dilution). Cells were then washed and incubated with the anti-Alexa Fluor 594 antibody. After washing, cells were incubated with DAPI for 30 min, washed again and then the cover slips were mounted on the slides. Confocal microscopy was performed using an Olympus IX- 81 Spinning Disc Confocal microscope using a 60x water immersion 1.2 NA objective and 31 Slidebook 5.0 software. Copy-Number-Variation Analysis
[0095] Copy-number-variation analysis of PX's tumor was conducted using a human genome CGH microarray kit (244A; Agilent Technologies). This platform uses 240,000 unique 60-mer oligonucleotide probes across the genome, with tighter coverage in the regions of RefSeq genes. Array hybridization was performed using 2.5 μg of gDNA from PX's tissue specimen and a control human gDNA (Promega) according to the array manufacturer's protocol. Briefly, hybridization lasted for 40 h at 65°C, and hybridized arrays were scanned using an Agilent Technologies dual-laser-based scanner. Data transformation was performed using the Feature Extraction CGH-v4_91 software program (Agilent Technologies). Statistical aberration detection was conducted using the Nexus Copy Number software program (version 5.0; BioDiscovery, El Segundo, CA).
MassARRAY Mutation Analysis
[0096] gDNA from the specimens was analyzed using polymerase chain reaction (PCR) amplification followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectroscopy single-nucleotide polymorphism analysis (MassARRAY; Sequenom, Inc., San Diego, CA) for 40 genes with 240 mutations (Table 1). PCR products were classified as
wt or mutant according to their molecular weights. Detected mutations were then Sanger sequenced.
Primers for Mutational Analysis of BRAF and c-Src
[0097] The primers used for c-Src were CTT CTC CTT TCC TCC CTC CTT (forward; SEQ ID NO:9) and CAG GAG AGG CAC TCT GCAC (reverse; SEQ ID NO: 10) for exon 7, AGC CAT ATC CAG GGA GAA GC (forward; SEQ ID NO: l 1) and ACA CCC AGC TCA AAC CAC TC (reverse; SEQ ID NO: 12) for exon 8, CCT TCC CTC CAA TGT CAG G (forward; SEQ ID NO: 13) and AGT CTG CAG CTG AGG CTT TG (reverse; SEQ ID NO: 14) for exon 9, and AGA AGA CCC GCC TAA CTG CT (forward; SEQ ID NO: 15) and ATC CAG CAG AGG CAG CTA AAG (reverse; SEQ ID NO: 16) for exon 10. The primers used for BRAF were TCC CTC TCA GGC ATA AGG TAA (forward; SEQ ID NO: 17) and CGA ACA GTG AAT ATT TCC TTT GAT (reverse; SEQ ID NO: 18) for exon 11 and TCA TAA TGC TTG CTC TGA TAG GA (forward; SEQ ID NO: 19) and GGC CAA AAA TTT AAT CAG TGG A (reverse; SEQ ID NO:20) for exon 15. Mutational Analysis of BRAF and c-Src
[0098] Intron-based PCR was used to sequence B-Raf exons 1 1 and 15 and c-Src exons 7-10 as described previously. FFPE tumor tissue was microdissected, and about 200 cells were used in each amplification. DNA extracted from either microdissected tissue or cell pellets was subjected to PCR, and PCR products were directly sequenced using a PRISM dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). The primers are listed in the supplemental methods. Sequence variants were confirmed using independent PCR amplifications and sequenced in both directions
DDR2 Sequencing
[0099] DDR2 mutations within coding exons were screened from genomic DNA obtained from patient's tumor sample using Sanger sequencing method as described (6). The coding exons were designated based on mRNA transcript variant 2 from DDR2 gene (NM_ 006182). Mutations( based on SNP locations) were verified manually with comparison made to the matched normal sequence.
Fluorescence In Situ Hybridization
[00100] A fluorescence in situ hybridization (FISH) assay following a standard protocol was performed to determine the presence of an ALK gene rearrangement using the LSI ALK dual-color break-apart probe (Abbott Molecular, Abbott Park, IL). PX's tissue slides were incubated for 4 hours at 56°C, deparaffinized in Citri-Solv (Fisher), and washed in 100% ethanol for 5 min. The slides were pretreated using a Dako histology FISH accessory kit (K5599) in a pressure cooker as follows: at 121°C for 1 min, cool-down to 90°C, then at room temperature for 15 min. Excess liquid was removed and the slides were incubated in Dako pepsin at 37°C for 2 hours. Next, the slides were washed in Dako wash buffer and dehydrated in ethanol. The probe sets were applied to the tissue which was covered with a coverslip and sealed with rubber cement. The specimen was denatured for 8 min at 80°C and incubated at 37°C for 48 h. Posthybridization washes were performed using a wash buffer (1.5 M urea in O. lx standard sodium citrate). The slides were then washed in 2x standard sodium citrate for 2 min and dehydrated in ethanol. Chromatin was counterstained with DAPI (0.3 μg/mL in Vectashield; Vector Laboratories, Burlingame, CA). Analysis was performed using a fluorescence microscope with single interference filter sets for green (SpectrumGreen), orange (SpectrumOrange), and blue (DAPI) bandpass filters. Monochromatic images were captured and merged using a CytoVision workstation (Applied Imaging, San Jose, CA). Cells with single or split signals were defined as those with gene rearrangement. Those with both signals close or overlapping were negative for rearrangement.
Genomic DNA Preparation
[00101] Total nucleic acids were extracted from five tissue sections using an SPRITE total nucleic acid extractor (Beckman Coulter). Each section was individually extracted and gDNA isolated. Briefly, sections were incubated in 200 of lysis buffer for 1 hour at 85 °C followed by 30 of proteinase K for 1 hour at 55°C, producing a total nucleic acid fraction. Next, gDNA was isolated from the sections and cleaned for array comparative genomic hybridization (aCGH) and matrix-assisted laser desorption/ionization-time-of-flight mass spectroscopy single-nucleotide polymorphism analysis using a standard column cleanup with RNase treatment to remove RNA from the total nucleic acid fraction (DNeasy blood and tissue kit; QIAGEN) and adapted from an FFPE DNA preparation for oligonucleotide array- based CGH for a gDNA analysis protocol (Agilent Technologies). gDNA quantity and purity
were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific). The mean gDNA quantity recovered per tissue section was 10,340 ng. The mean sample purity (1.81) as assessed using a 260:280 wavelength ratio was within accepted DNA-purity levels (28). Individual gDNA preparations from each section were then pooled for use. Immunohistochemistry
[00102] An automated stainer (Dako) was used for immunohistochemical staining of tumor sections. Five-micrometer sections of FFPE tumor were deparaffinized and hydrated, and antigen retrieval was done (pH 6.0, Dako). Cell pellets from NSCLC cell lines with high and low protein expression levels according to Western blotting relative to the levels in a panel of eight NSCLC cell lines were used as positive and negative controls for the staining. Peroxide blocking was performed using 3% methanol and hydrogen peroxide for 15 min. Upon blocking with 10% fetal bovine serum, tissue slides were incubated with the primary antibodies phospho-PDGFRa (Y754, 1 :500), total PDGFRa (1 :500), c-Kit (1 :200), EphA2 (1 :500), phospho-EphA2 (Tyr594, 1 :500), phospho-c-Kit (Y719, 1 : 150), and p53 (1 :400) at room temperature for 1 hour. After washing in Tris-buffered saline and Tween 20, slides were incubated with Dako EnVision+ Dual Link reagent for 30 min at room temperature followed by a Dako chromogen substrate for 5 min and were then counterstained with hematoxylin for 5 min. Slides were examined for the staining intensity by a blinded observer using a light microscope with a *20 magnification objective. Cytoplasmic staining was scored based on both staining intensity (0-3 scale: 0, below the level of detection; 1 , weak; 2, moderate; 3, strong) and the percentage of cells stained at each intensity level (0- 100%). The final score was calculated by multiplying the intensity score by the percentage, producing a scoring range of 0-300.
* * * [00103] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the
agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
1. T. J. Lynch, D. W. Bell, R. Sordella, S. Gurubhagavatula, R. A. Okimoto, B. W.
Brannigan, P. L. Harris, S. M. Haserlat, J. G. Supko, F. G. Haluska, D. N. Louis, D.
C. Christiani, J. Settleman, and D. A. Haber,Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non-Small-Cell Lung Cancer to Gefitinib. N Engl J Med 350, 2129-2139 (2004).
2. F. M. Johnson, B. N. Bekele, L. Feng, I. Wistuba, X. M. Tang, H. T. Tran, J. J.
Erasmus, L. L. Hwang, N. Takebe, G. R. Blumenschein, S. M. Lippman, and D. J. Stewart,Phase Ii Study of Dasatinib in Patients with Advanced Non-Small-Cell Lung Cancer. J Clin Oncol 28, 4609-4615 (2010).
3. T. Kuilman, C. Michaloglou, W. J. Mooi, and D. S. Peeper, The Essence of Senescence. Genes Dev 24, 2463-2479 (2010).
4. S. J. Heidorn, C. Milagre, S. Whittaker, A. Nourry, I. Niculescu-Duvas, N. Dhomen, J. Hussain, J. S. Reis-Filho, C. J. Springer, C. Pritchard, and R. Marais,Kinase-Dead Braf and Oncogenic Ras Cooperate to Drive Tumor Progression through Craf. Cell
140, 209-221 (2010).
5. P. I. Poulikakos, C. Zhang, G. Bollag, K. M. Shokat, and N. Rosen,Raf Inhibitors Transactivate Raf Dimers and Erk Signalling in Cells with Wild-Type Braf. Nature 464, 427-430 (2010).
6. P. S. Hammerman, M. L. Sos, A. H. Ramos, C. Xu, A. Dutt, W. Zhou, L. E. Brace, B.
A. Woods, W. Lin, J. Zhang, X. Deng, S. M. Lim, S. Heynck, M. Peifer, J. R. Simard, M. S. Lawrence, R. C. Onofrio, H. B. Salvesen, D. Seidel, T. Zander, J. M. Heuckmann, A. Soltermann, H. Moch, M. Koker, F. Leenders, F. Gabler, S. Querings, S. AnsA©n, E. Brambilla, C. Brambilla, P. Lorimier, O. T. Brustugun, A. s. Helland, I. Petersen, J. H. Clement, H. Groen, W. Timens, H. Sietsma, E. Stoelben, J. r. Wolf,
D. G. Beer, M. S. Tsao, M. Hanna, C. Hatton, M. J. Eck, P. A. Janne, B. E. Johnson, W. Winckler, H. Greulich, A. J. Bass, J. Cho, D. Rauh, N. S. Gray, K.-K. Wong, E. B. Haura, R. K. Thomas, and M. Meyerson,Mutations in the Ddr2 Kinase Gene Identify
a Novel Therapeutic Target in Squamous Cell Lung Cancer. Cancer Discovery 1, 0F76-70F87 (2011).
M. L. Sos, K. Michel, T. Zander, J. Weiss, P. Frommolt, M. Peifer, D. Li, R. Ullrich, M. Koker, F. Fischer, T. Shimamura, D. Rauh, C. Mermel, S. Fischer, I. Stuckrath, S. Heynck, R. Beroukhim, W. Lin, W. Winckler, K. Shah, T. LaFramboise, W. F. Moriarty, M. Hanna, L. Tolosi, J. Rahnenfuhrer, R. Verhaak, D. Chiang, G. Getz, M. Hellmich, J. Wolf, L. Girard, M. Peyton, B. A. Weir, T. H. Chen, H. Greulich, J. Barretina, G. I. Shapiro, L. A. Garraway, A. F. Gazdar, J. D. Minna, M. Meyerson, K. K. Wong, and R. K. Thomas,Predicting Drug Susceptibility of Non-Small Cell Lung Cancers Based on Genetic Lesions. J Clin Invest 1 19, 1727-1740 (2009).
K. S. Smalley, M. Xiao, J. Villanueva, T. K. Nguyen, K. T. Flaherty, R. Letrero, P. Van Belle, D. E. Elder, Y. Wang, K. L. Nathanson, and M. Herlyn,Craf Inhibition Induces Apoptosis in Melanoma Cells with Non-V600e Braf Mutations. Oncogene 28, 85-94 (2009).
J. Li, U. Rix, B. Fang, Y. Bai, A. Edwards, J. Colinge, K. L. Bennett, J. Gao, L. Song, S. Eschrich, G. Superti-Furga, J. Koomen, and E. B. Haura,A Chemical and Phosphoproteomic Characterization of Dasatinib Action in Lung Cancer. Nat Chem Biol 6, 291-299 (2010).
L. M. Packer, S. Rana, R. Hayward, T. O'Hare, C. A. Eide, A. Rebocho, S. Heidorn, M. S. Zabriskie, I. Niculescu-Duvaz, B. J. Druker, C. Springer, and R. Marais,Nilotinib and Mek Inhibitors Induce Synthetic Lethality through Paradoxical Activation of Raf in Drug-Resistant Chronic Myeloid Leukemia. Cancer Cell 20, 715- 727 (2012).
M. Collado, and M. Serrano,Senescence in Tumours: Evidence from Mice and Humans. Nat Rev Cancer 10, 51-57 (2010).
P. I. Poulikakos, Y. Persaud, M. Janakiraman, X. Kong, C. Ng, G. Moriceau, H. Shi, M. Atefi, B. Titz, M. T. Gabay, M. Saltan, K. B. Dahlman, M. Tadi, J. A. Wargo, K. T. Flaherty, M. C. Kelley, T. Misteli, P. B. Chapman, J. A. Sosman, T. G. Graeber, A. Ribas, R. S. Lo, N. Rosen, and D. B. Solit,Raf Inhibitor Resistance Is Mediated by Dimerization of Aberrantly Spliced Braf(V600e). Nature 480, 387-390 (2011).
E. U. Alejandro, T. B. Kalynyak, F. Taghizadeh, K. S. Gwiazda, E. K. Rawstron, K. J. Jacob, and J. D. Johnson,Acute Insulin Signaling in Pancreatic Beta-Cells Is Mediated by Multiple Raf-1 Dependent Pathways. Endocrinology 151, 502-512 (2010).
A. Mielgo, L. Seguin, M. Huang, M. F. Camargo, S. Anand, A. Franovic, S. M. Weis, S. J. Advani, E. A. Murphy, and D. A. Cheresh,A Mek-Independent Role for Craf in Mitosis and Tumor Progression. Nat Med 17, 1641-1645 (2011).
B. Sen, S. Peng, B. Saigal, M. D. Williams, and F. M. Johnson,Distinct Interactions between C-Src and C-Met in Mediating Resistance to C-Src Inhibition in Head and Neck Cancer. Clin Cancer Res 17, 514-524 (2011).
F. M. Johnson, B. Saigal, M. Talpaz, and N. J. Donato,Dasatinib (Bms-354825) Tyrosine Kinase Inhibitor Suppresses Invasion and Induces Cell Cycle Arrest and Apoptosis of Head and Neck Squamous Cell Carcinoma and Non-Small Cell Lung Cancer Cells. Clin Cancer Res 11, 6924-6932 (2005).
L. N. Kwong, and L. Chin,The Brothers Raf. Cell 140, 180-182 (2010).
K. T. Flaherty, and G. McArthur,Braf, a Target in Melanoma: Implications for Solid Tumor Drug Development. Cancer 116, 4902-4913 (2010).
A. Marchetti, L. Felicioni, S. Malatesta, M. G. Sciarrotta, L. Guetti, A. Chella, P. Viola, C. Pullara, F. Mucilli, and F. Buttitta,Clinical Features and Outcome of Patients with Non-Small-Cell Lung Cancer Harboring Braf Mutations. J Clin Oncol, (201 1).
P. K. Paik, M. E. Arcila, M. Fara, C. S. Sima, V. A. Miller, M. G. Kris, M. Ladanyi, and G. J. Riely,Clinical Characteristics of Patients with Lung Adenocarcinomas Harboring Braf Mutations. J Clin Oncol, (2011).
V. Emuss, M. Garnett, C. Mason, and R. Marais, Mutations of C-Raf Are Rare in Human Cancer Because C-Raf Has a Low Basal Kinase Activity Compared with B- Raf. Cancer Res 65, 9719-9726 (2005).
E. W. Joseph, C. A. Pratilas, P. I. Poulikakos, M. Tadi, W. Wang, B. S. Taylor, E. Halilovic, Y. Persaud, F. Xing, A. Viale, J. Tsai, P. B. Chapman, G. Bollag, D. B. Solit, and N. Rosen,The Raf Inhibitor Plx4032 Inhibits Erk Signaling and Tumor Cell Proliferation in a V600e Braf-Selective Manner. Proc Natl Acad Sci U S A 107, 14903-14908 (2010).
L. K. Rushworth, A. D. Hindley, E. O'Neill, and W. Kolch,Regulation and Role of Raf-l/B-Raf Heterodimerization. Mol Cell Biol 26, 2262-2272 (2006).
O. E. Sturm, R. Orton, J. Grindlay, M. Birtwistle, V. Vyshemirsky, D. Gilbert, M. Calder, A. Pitt, B. Kholodenko, and W. Kolch,The Mammalian Mapk/Erk Pathway Exhibits Properties of a Negative Feedback Amplifier. Sci Signal 3, ra90 (2010).
T. S. Niault, and M. Baccarini,Targets of Raf in Tumorigenesis. Carcinogenesis 31, 1165-1 174 (2010).
K. T. Flaherty, I. Puzanov, K. B. Kim, A. Ribas, G. A. McArthur, J. A. Sosman, P. J.
O'Dwyer, R. J. Lee, J. F. Grippo, K. Nolop, and P. B. Chapman,Inhibition of Mutated,
Activated Braf in Metastatic Melanoma. N Engl J Med 363, 809-819 (2010).
T. Kamata, J. Hussain, S. Giblett, R. Hayward, R. Marais, and C. Pritchard,Braf
Inactivation Drives Aneuploidy by Deregulating Craf. Cancer Res 70, 8475-8486
(201 1).
R. B. Blasco, S. Francoz, D. Santamaria, M. Canamero, P. Dubus, J. Charron, M. Baccarini, and M. Barbacid,C-Raf, but Not B-Raf, Is Essential for Development of K- Ras Oncogene-Driven Non-Small Cell Lung Carcinoma. Cancer Cell 19, 652-663 (201 1).
L. M. Fedorov, T. Papadopoulos, O. Y. Tyrsin, T. Twardzik, R. Gotz, and U. R. Rapp,Loss of P53 in Craf-Induced Transgenic Lung Adenoma Leads to Tumor Acceleration and Phenotypic Switch. Cancer Res 63, 2268-2277 (2003).
H. G. Kim, S. Y. Hwang, S. A. Aaronson, A. Mandinova, and S. W. Lee,Ddrl Receptor Tyrosine Kinase Promotes Prosurvival Pathway through Notchl Activation. J Biol Chem 286, 17672-17681 (201 1).
B. Sen, B. Saigal, N. Parikh, G. Gallick, and F. M. Johns on, Sustained Src Inhibition Results in Signal Transducer and Activator of Transcription 3 (Stat3) Activation and Cancer Cell Survival Via Altered Janus -Activated Kinase-Stat3 Binding. Cancer Res 69, 1958-1965 (2009).
T. C. Chou, and P. Talalay,Quantitative Analysis of Dose-Effect Relationships: The Combined Effects of Multiple Drugs or Enzyme Inhibitors. Adv Enzyme Regul 22, 27-55 (1984).
P. T. Wan, M. J. Garnett, S. M. Roe, S. Lee, D. Niculescu-Duvaz, V. M. Good, C. M. Jones, C. J. Marshall, C. J. Springer, D. Barford, and R. Marais,Mechanism of Activation of the Raf-Erk Signaling Pathway by Oncogenic Mutations of B-Raf. Cell 116, 855-867 (2004).
T. Ikenoue, Y. Hikiba, F. Kanai, Y. Tanaka, J. Imamura, T. Imamura, M. Ohta, H. Ijichi, K. Tateishi, T. Kawakami, J. Aragaki, M. Matsumura, T. Kawabe, and M. Omata,Functional Analysis of Mutations within the Kinase Activation Segment of B- Raf in Human Colorectal Tumors. Cancer Res 63, 8132-8137 (2003).
Claims
1. An in vitro method for identifying a candidate for a Src inhibitor therapy comprising:
(a) testing a biological sample from a subject having a cancer to determine whether cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity relative to a wild type BRAF protein; and
(b) identifying the subject as a good candidate for a Src inhibitor therapy if the sample from the subject indicates that cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity; or identifying the subject as a poor candidate for a Src inhibitor therapy if the sample from the subject does not indicates that cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity.
2. A method for identifying a good candidate for a Src inhibitor therapy comprising testing a biological sample from a subject having a cancer to determine whether cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity relative to a wild type BRAF protein, thereby identifying the subject as a good candidate for a Src inhibitor therapy.
3. The method of claim 1, wherein testing the sample comprises determining whether cells of the cancer comprises a BRAF gene encoding a protein with reduced kinase activity.
4. The method of claim 3, wherein the BRAF gene encodes a protein comprising an amino acid substitution or deletion at position Y472.
5. The method of claim 4, wherein the BRAF gene encodes a protein comprising a Y472C amino acid substitution.
6. The method of claim 1, wherein testing the sample from the subject comprises determining all or part of a BRAF gene sequence in the sample.
7. The method of claim 1, wherein testing the sample from the subject comprises measuring BRAF kinase activity in the sample.
8. The method of claim 1, wherein the sample comprises cancer cells.
9. The method of claim 1, wherein a good candidate for a Src inhibitor therapy comprises a subject that is predicted to have reduced in tumor size or burden, reduced tumor growth, reduced tumor-associated pain, reduced cancer associated pathology, reduced cancer associated symptoms, cancer non-progression, cancer remission, reduced cancer metastasis, or increased survival time in response to a Src inhibitor therapy.
10. The method of claim 1, wherein the cancer is a lung cancer.
11. The method of claim 10, wherein the lung cancer is a non-small cell lung cancer.
12. The method of claim 10, wherein the cancer is a metastatic lung cancer.
13. The method of claim 1 or 2, wherein the Src inhibitor therapy is a Src kinase inhibitor.
14. The method of claim 13, wherein the Src kinase inhibitor is bosutinib, saracatinib, danusertib, NVP-BHG712, quercetin, PCI-32765, KX2-391, AP23451 or dasatinib.
15. The method of claim 14, wherein the Src kinase inhibitor is dasatinib.
16. The method of claim 1, further comprising providing a report indicating whether the subject is a good candidate or a poor candidate for a Src inhibitor therapy.
17. The method of claim 16, wherein the report is a written or electronic report.
18. The method of claim 16, wherein providing a report comprises transmitting the report to a doctor or a hospital.
19. A method for treating a subject having a cancer, wherein it was determined that cancer cells from the subject comprise reduced BRAF kinase activity relative to a control level, the method comprising administering a Src inhibitor therapy to the subject.
20. The method of claim 19, wherein the cancer is a lung cancer.
21. The method of claim 20, wherein the lung cancer is a non-small cell lung cancer.
22. The method of claim 20, wherein the cancer is a metastatic lung cancer.
23. The method of claim 19, wherein the Src inhibitor therapy is a Src kinase inhibitor.
24. The method of claim 23, wherein the Src kinase inhibitor is bosutinib, saracatinib, danusertib, NVP-BHG712, quercetin, PCI-32765, KX2-391, AP23451 or dasatinib.
25. The method of claim 24, wherein the Src kinase inhibitor is dasatinib.
26. The method of claim 19, wherein it was determined that cancer cells from the subject comprise a BRAF gene encoding a protein with reduced kinase activity.
27. The method of claim 26, wherein the BRAF gene encodes a protein comprising an amino acid substitution or deletion at position Y472.
28. The method of claim 27, wherein the BRAF gene encodes a protein comprising a Y472C amino acid substitution.
29. The method of claim 19, wherein the Src inhibitor therapy is administer two or more times.
30. The method of claim 19, further comprising administering at least a second anticancer therapy to the subject.
31. The method of claim 30, wherein the second anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy or cytokine therapy.
32. A composition for the treatment of a subject having cancer comprising cancer cells with reduced BRAF kinase activity relative to a control level, the composition comprising a therapeutically effective amount of a Src inhibitor.
33. A tangible computer-readable medium comprising computer-readable code that, when executed by a computer, causes the computer to perform operations comprising:
a) receiving information corresponding to a BRAF kinase activity in a sample from a subject having a cancer; and
b) determining a relative level of BRAF kinase activity compared to a reference level, wherein reduced BRAF kinase activity relative to the reference level indicates the subject is a good candidate for a Src inhibitor therapy.
34. The method of claim 33, wherein the information corresponding to a BRAF kinase activity comprises all or part of a BRAF gene sequence.
35. The method of claim 34, wherein determining a relative level of BRAF kinase activity comprises determining a change in all or part of a BRAF gene sequence compared to a reference sequence, wherein a change in the BRAF gene sequence corresponding to reduced BRAF activity indicates that the subject is a good candidate for a Src inhibitor therapy.
36. The method of claim 34, wherein the reference level is all or part of a wild type BRAF gene sequence.
37. The tangible computer-readable medium of claim 33, wherein the receiving information comprises receiving from a tangible data storage device information corresponding to a level of BRAF kinase activity in a sample from a subject having a cancer.
38. The tangible computer-readable medium of claim 33, further comprising computer- readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising: sending information corresponding to the relative level of BRAF kinase activity to a tangible data storage device.
39. The tangible computer-readable medium of claim 33, wherein the computer-readable code that, when executed by a computer, causes the computer to perform operations further comprising: c) calculating a diagnostic score for the sample, wherein the diagnostic score is indicative of the probability that the subject will respond to a Src inhibitor therapy.
40. A method for treating a subject having a cancer comprising administering to the subject an effective amount of a Src inhibitor in conjunction with a RAF inhibitor.
41. The method of claim 40, wherein the Src inhibitor is a Src kinase inhibitor.
42. The method of claim 41, wherein the Src kinase inhibitor is bosutinib, saracatinib, danusertib, NVP-BHG712, quercetin, PCI-32765, KX2-391, AP23451 or dasatinib.
43. The method of claim 42, wherein the Src kinase inhibitor is dasatinib.
44. The method of claim 40, wherein the RAF inhibitor is a BRAF specific inhibitor
45. The method of claim 40, wherein the RAF inhibitor is vemurafenib, GSK2118436 or sorafenib.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201261621747P | 2012-04-09 | 2012-04-09 | |
US61/621,747 | 2012-04-09 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2013155077A1 true WO2013155077A1 (en) | 2013-10-17 |
Family
ID=49328096
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2013/035783 WO2013155077A1 (en) | 2012-04-09 | 2013-04-09 | Response markers for src inhibitor therapies |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2013155077A1 (en) |
Cited By (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110291403A (en) * | 2016-12-20 | 2019-09-27 | 特里特福尔莱弗公司 | Pass through the method for mass spectrometric determination BRAF mutation and wild type BRAF albumen |
US10646488B2 (en) | 2016-07-13 | 2020-05-12 | Araxes Pharma Llc | Conjugates of cereblon binding compounds and G12C mutant KRAS, HRAS or NRAS protein modulating compounds and methods of use thereof |
US10647703B2 (en) | 2015-09-28 | 2020-05-12 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
TWI694837B (en) * | 2014-09-18 | 2020-06-01 | 美商亞瑞克西斯製藥公司 | Combination therapies for treatment of cancer |
US10689356B2 (en) | 2015-09-28 | 2020-06-23 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
US10723738B2 (en) | 2016-09-29 | 2020-07-28 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
US10730867B2 (en) | 2015-09-28 | 2020-08-04 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
US10736897B2 (en) | 2017-05-25 | 2020-08-11 | Araxes Pharma Llc | Compounds and methods of use thereof for treatment of cancer |
US10829458B2 (en) | 2015-04-10 | 2020-11-10 | Araxes Pharma Llc | Substituted quinazoline compounds and methods of use thereof |
US10858343B2 (en) | 2015-09-28 | 2020-12-08 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
US10882847B2 (en) | 2015-09-28 | 2021-01-05 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
US10919850B2 (en) | 2013-03-15 | 2021-02-16 | Araxes Pharma Llc | Covalent inhibitors of KRas G12C |
US11021470B2 (en) | 2015-11-16 | 2021-06-01 | Araxes Pharma Llc | 2-substituted quinazoline compounds comprising a substituted heterocyclic group and methods of use thereof |
US11040027B2 (en) | 2017-01-17 | 2021-06-22 | Heparegenix Gmbh | Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death |
US11059819B2 (en) | 2017-01-26 | 2021-07-13 | Janssen Biotech, Inc. | Fused hetero-hetero bicyclic compounds and methods of use thereof |
US11136308B2 (en) | 2017-01-26 | 2021-10-05 | Araxes Pharma Llc | Substituted quinazoline and quinazolinone compounds and methods of use thereof |
US11274093B2 (en) | 2017-01-26 | 2022-03-15 | Araxes Pharma Llc | Fused bicyclic benzoheteroaromatic compounds and methods of use thereof |
US11279689B2 (en) | 2017-01-26 | 2022-03-22 | Araxes Pharma Llc | 1-(3-(6-(3-hydroxynaphthalen-1-yl)benzofuran-2-yl)azetidin-1 yl)prop-2-en-1-one derivatives and similar compounds as KRAS G12C modulators for treating cancer |
US11351167B2 (en) | 2014-01-28 | 2022-06-07 | Buck Institute For Research On Aging | Treating cognitive decline and other neurodegenerative conditions by selectively removing senescent cells from neurological tissue |
US11358959B2 (en) | 2017-01-26 | 2022-06-14 | Araxes Pharma Llc | Benzothiophene and benzothiazole compounds and methods of use thereof |
US11377441B2 (en) | 2017-05-25 | 2022-07-05 | Araxes Pharma Llc | Covalent inhibitors of KRAS |
US11517572B2 (en) * | 2014-01-28 | 2022-12-06 | Mayo Foundation For Medical Education And Research | Killing senescent cells and treating senescence-associated conditions using a SRC inhibitor and a flavonoid |
US11639346B2 (en) | 2017-05-25 | 2023-05-02 | Araxes Pharma Llc | Quinazoline derivatives as modulators of mutant KRAS, HRAS or NRAS |
US11878985B2 (en) | 2013-10-10 | 2024-01-23 | Araxes Pharma Llc | Substituted quinazolines as inhibitors of KRAS G12C |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7901876B2 (en) * | 2004-09-30 | 2011-03-08 | Ifom Fondazione Instituto Firc Di Oncologia Molecolare | Cancer markers |
WO2011054030A1 (en) * | 2009-11-05 | 2011-05-12 | Cephalon Australia Pty Ltd | Treatment of cancer involving mutated kras or braf genes |
US20110319415A1 (en) * | 2008-08-18 | 2011-12-29 | Universit+e,uml a+ee t zu K+e,uml o+ee ln | Susceptibility to HSP90-Inhibitors |
-
2013
- 2013-04-09 WO PCT/US2013/035783 patent/WO2013155077A1/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7901876B2 (en) * | 2004-09-30 | 2011-03-08 | Ifom Fondazione Instituto Firc Di Oncologia Molecolare | Cancer markers |
US20110319415A1 (en) * | 2008-08-18 | 2011-12-29 | Universit+e,uml a+ee t zu K+e,uml o+ee ln | Susceptibility to HSP90-Inhibitors |
WO2011054030A1 (en) * | 2009-11-05 | 2011-05-12 | Cephalon Australia Pty Ltd | Treatment of cancer involving mutated kras or braf genes |
Non-Patent Citations (2)
Title |
---|
CARONIA ET AL.: "Role of BRAF in thyroid oncogenesis", CLINICAL CANCER RESEARCH, vol. 17, no. 24, 15 December 2011 (2011-12-15) * |
SEN ET AL.: "Kinase-impaired BRAF mutations in lung cancer confer sensitivity to dasatinib", SCIENCE TRANSLATIONAL MEDICINE, vol. 4, no. 136, 30 May 2012 (2012-05-30) * |
Cited By (27)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10919850B2 (en) | 2013-03-15 | 2021-02-16 | Araxes Pharma Llc | Covalent inhibitors of KRas G12C |
US11878985B2 (en) | 2013-10-10 | 2024-01-23 | Araxes Pharma Llc | Substituted quinazolines as inhibitors of KRAS G12C |
US11980616B2 (en) | 2014-01-28 | 2024-05-14 | Mayo Foundation For Medical Education And Research | Treating liver disease by selectively eliminating senescent cells |
US11963957B2 (en) | 2014-01-28 | 2024-04-23 | Mayo Foundation For Medical Education And Research | Treating cardiovascular disease by selectively eliminating senescent cells |
US11517572B2 (en) * | 2014-01-28 | 2022-12-06 | Mayo Foundation For Medical Education And Research | Killing senescent cells and treating senescence-associated conditions using a SRC inhibitor and a flavonoid |
US11351167B2 (en) | 2014-01-28 | 2022-06-07 | Buck Institute For Research On Aging | Treating cognitive decline and other neurodegenerative conditions by selectively removing senescent cells from neurological tissue |
TWI694837B (en) * | 2014-09-18 | 2020-06-01 | 美商亞瑞克西斯製藥公司 | Combination therapies for treatment of cancer |
US10829458B2 (en) | 2015-04-10 | 2020-11-10 | Araxes Pharma Llc | Substituted quinazoline compounds and methods of use thereof |
US10730867B2 (en) | 2015-09-28 | 2020-08-04 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
US10882847B2 (en) | 2015-09-28 | 2021-01-05 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
US10858343B2 (en) | 2015-09-28 | 2020-12-08 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
US10647703B2 (en) | 2015-09-28 | 2020-05-12 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
US10689356B2 (en) | 2015-09-28 | 2020-06-23 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
US11021470B2 (en) | 2015-11-16 | 2021-06-01 | Araxes Pharma Llc | 2-substituted quinazoline compounds comprising a substituted heterocyclic group and methods of use thereof |
US10646488B2 (en) | 2016-07-13 | 2020-05-12 | Araxes Pharma Llc | Conjugates of cereblon binding compounds and G12C mutant KRAS, HRAS or NRAS protein modulating compounds and methods of use thereof |
US10723738B2 (en) | 2016-09-29 | 2020-07-28 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
CN110291403A (en) * | 2016-12-20 | 2019-09-27 | 特里特福尔莱弗公司 | Pass through the method for mass spectrometric determination BRAF mutation and wild type BRAF albumen |
CN110291403B (en) * | 2016-12-20 | 2022-09-16 | 特里特福尔莱弗公司 | Method for determining BRAF mutations and wild-type BRAF proteins by mass spectrometry |
US11040027B2 (en) | 2017-01-17 | 2021-06-22 | Heparegenix Gmbh | Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death |
US11358959B2 (en) | 2017-01-26 | 2022-06-14 | Araxes Pharma Llc | Benzothiophene and benzothiazole compounds and methods of use thereof |
US11279689B2 (en) | 2017-01-26 | 2022-03-22 | Araxes Pharma Llc | 1-(3-(6-(3-hydroxynaphthalen-1-yl)benzofuran-2-yl)azetidin-1 yl)prop-2-en-1-one derivatives and similar compounds as KRAS G12C modulators for treating cancer |
US11274093B2 (en) | 2017-01-26 | 2022-03-15 | Araxes Pharma Llc | Fused bicyclic benzoheteroaromatic compounds and methods of use thereof |
US11136308B2 (en) | 2017-01-26 | 2021-10-05 | Araxes Pharma Llc | Substituted quinazoline and quinazolinone compounds and methods of use thereof |
US11059819B2 (en) | 2017-01-26 | 2021-07-13 | Janssen Biotech, Inc. | Fused hetero-hetero bicyclic compounds and methods of use thereof |
US11377441B2 (en) | 2017-05-25 | 2022-07-05 | Araxes Pharma Llc | Covalent inhibitors of KRAS |
US11639346B2 (en) | 2017-05-25 | 2023-05-02 | Araxes Pharma Llc | Quinazoline derivatives as modulators of mutant KRAS, HRAS or NRAS |
US10736897B2 (en) | 2017-05-25 | 2020-08-11 | Araxes Pharma Llc | Compounds and methods of use thereof for treatment of cancer |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2013155077A1 (en) | Response markers for src inhibitor therapies | |
Stucklin et al. | Alterations in ALK/ROS1/NTRK/MET drive a group of infantile hemispheric gliomas | |
Li et al. | RET fusions in solid tumors | |
Mondal et al. | Pediatric bithalamic gliomas have a distinct epigenetic signature and frequent EGFR exon 20 insertions resulting in potential sensitivity to targeted kinase inhibition | |
Long et al. | Increased MAPK reactivation in early resistance to dabrafenib/trametinib combination therapy of BRAF-mutant metastatic melanoma | |
Axelrod et al. | Axl as a mediator of cellular growth and survival | |
Lee et al. | Somatic mutations in epidermal growth factor receptor signaling pathway genes in non-small cell lung cancers | |
EP2740742B1 (en) | Fusion gene of kif5b gene and ret gene, and method for determining effectiveness of cancer treatment targeting fusion gene | |
Gerber et al. | ALK inhibition for non-small cell lung cancer: from discovery to therapy in record time | |
US10421820B2 (en) | Bruton's tyrosine kinase as anti-cancer drug target | |
Yang et al. | Overcoming erlotinib resistance with tailored treatment regimen in patient‐derived xenografts from naïve Asian NSCLC patients | |
All-Ericsson et al. | c-Kit–dependent growth of uveal melanoma cells: a potential therapeutic target? | |
JP2020202839A (en) | Biomarker for predicting sensitivity to protein kinase inhibitor and use thereof | |
US9283244B2 (en) | Treatment of cancer by inhibiting activity or expression of late SV-40 factor | |
EP3020828A1 (en) | Method of predicting response of cancer to treatment | |
US20160032399A1 (en) | Method for the Prognosis and Treatment of Renal Cell Carcinoma Metastasis | |
Wang et al. | Bruton’s tyrosine kinase and its isoforms in cancer | |
Ning et al. | Identification of anaplastic lymphoma kinase as a potential therapeutic target in Basal Cell Carcinoma | |
Turrell et al. | Age-associated microenvironmental changes highlight the role of PDGF-C in ER+ breast cancer metastatic relapse | |
WO2019111998A1 (en) | Cancer spheroid production method and method for selecting colon cancer patients | |
da Silva et al. | Salivary glands adenoid cystic carcinoma: a molecular profile update and potential implications | |
Satgunaseelan et al. | Oral squamous cell carcinoma in young patients show higher rates of EGFR amplification: implications for novel personalized therapy | |
JP6858563B2 (en) | Prediction of EGFR inhibitor effect by BRAF mutation detection | |
KR101775356B1 (en) | Method for Determining Susceptibility to Dual Inhibitor against PARP and Tankyrase | |
Schweppe | Thyroid cancer cell lines: critical models to study thyroid cancer biology and new therapeutic targets |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 13775939 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 13775939 Country of ref document: EP Kind code of ref document: A1 |