Supplementary Table S1. Primary antibodies for Western blot analysis. Antibody name and specifications Manufacturer Actin (C-11) sc-1615, Santa Cruz, CA Akt1 (B-1) sc-5298 GAPDH (6C5) sc-32233 JAK2 (HR-758) sc-278 PDGFR-α (C-20) sc-338 phospho-pdgfr-α (Tyr 720) sc-12910-r Ron β (C-20) sc-322 p-ron β (Tyr1238/ Tyr1239) sc-16838 Raf-1 (E-10) sc-7267 Stat3 (F-2) sc-8019 Stat5 (C-17) sc-835 Stat6 (STAAD23A) sc-81387 Trk (C-14) sc-11 Phospho-MSPR/RON (Y1238/Y1239) AF1947 R&D Systems, Minneapolis, MN Caspase-3 #9662 Technology, Danvers, MA p44/42 MAPK (Erk1/2) (3A7) #9107 Phospho-Akt (Ser473) (D9E) #4060 Phospho-c-Raf (Ser338) (56A6) #9427 Phospho-Jak2 (Tyr1007/1008) #3771 Phoshpo-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) #4370 Phospho-Stat3 (Tyr705) #9131 Phospho-Stat5 (Tyr694) (C11C5) #9359 Phospho-TrkA (Tyr490) #9141 Phospho-Stat6 (Y641) #611566 BD Transduction Laboratories, San Jose, CA
Supplementary Table S2. Primer sequences for qrt-pcr. Primer name Primer sequence GADD153-for 5 -TGAGCGTATCATGTTAAAGATGAGCG-3 GADD153-rev 5 -GGTGTGGTGATGTATGAAGATACACTTCC-3 GADD34-for 5 -TGATCCGGACCCTGAGACTCC-3 GADD34-rev 5 -CCCAGACAGCCAGGAAATGG-3 LIPH-for 5 -TCTGGGTTTGTTGGAGAGATGTACG-3 LIPH-rev 5 -CTGGGATCTAATCTGTCTTGGTGAGG-3 PIM2-for 5 -CAAAGTGATTCCCCGGAATCG-3 PIM2rev 5 -ACCCACTTTCCATAGCAGTGCG-3 PPIA-for 5 -TGGACCCAACACAAATGGTTCC-3 PPIA-rev 5 -CATGGCCTCCACAATATTCATGC-3 SOS2-for 5 -CGAGGAGAACAGTCCGAAATGG-3 SOS2-rev 5 -AGAGAGACTCTTCATTAGCTGAGAGAGTGG-3 STAT4-for 5 -TGGCTGCAGCCAACATGC-3 STAT4-rev 5 -GCCACTTTGTGCTCCACATTCC-3 TNFSF10-for 5 -CAGTCTCTCTGTGTGGCTGTAACTTACG-3 TNFSF10-rev1 5 -CATCTTCTTTTAAGAAACAAGCAATGCC-3 TNFSF10-rev2 5 -AGCAATGACCCTGTCTGCTGC-3 TNFSF8-for 5 -TCTTCACGGTGGCCACTATTATGG-3 TNFSF8-rev 5 -TTCTGAGCAATTTCCTCCTTTGAGG-3 TRAF3IP3-for 5 -GACCTACTGTCCACTCTGATTCAGGC-3 TRAF3IP3-rev 5 -GGGTACAGGTGTATAGCTGGTTTTCTAAGG-3
Supplementary Table S3. TKIs and conventional chemotherapeutics used in this study. Compound Solvent Dasatinib (LC Laboratories, Woburn, MA) Doxorubicin (Merck Chemicals, Darmstadt, Germany) Erlotinib (LC Laboratories) Gefitinib (LC Laboratories) Lapatinib (LC Laboratories) Lestaurtinib (LC Laboratories) Sorafenib (LC Laboratories) Sunitinib (LC Laboratories) Vinblastine (Sigma-Aldrich, St. Louis, MO) Vandetanib (LC Laboratories) Vatalanib (LC Laboratories) H 2 O TKIs and conventional chemotherapeutics used in this study and their solvents. Supplementary Table S4. Sensitivity of Hodgkin-Reed/Sternberg cell lines to tyrosine kinase inhibitors (TKIs) at concentrations achievable in patients. % of viable cells TKI L-1236 L-428 KM-H2 Dasatinib 1 µm 48 h 106 - - Lapatinib 3 µm 72 h 81 90 84 Sunitinib 300 nm 72h 97 96 95 Percent of viable cells after treatment with TKIs dasatinib, lapatinib and sunitinib at concentrations achievable in patients. TKIs showing inhibitory effects on HRS cell lines in the initial screening at concentrations of 5 µm were applied at concentrations achievable in the plasma of patients and compared to a solvent treated control at optimal growth conditions. At the indicated achievable concentrations the TKIs did reduce the number of viable cells below 80%.
Supplementary Table S5. Amount of phosphorylated RTK in relation to total RTK amount. Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Figure 3A p-trk in L-1236 1 0.84 0.86 2.36 0.67 0.66 p-trk in HDLM-2 1 1.07 1.22 5.76 1.67 2.28 Figure 3B p-ron in L-428 1 0.4 0.25 Figure 3C p-pdgfrα in U-HO1 1 0.7 0.3 2.02 0.77 0.87 Figure 3D p-pdgfrα in KM-H2 1 8.12 0.22 Figure 3E p-pdgfrα in KM-H2 1 0.65 1 0.68 Densitometric analysis of Western blots shown in Figure 3. The relative phosphorylation of RTKs was normalized to the detected total amount of RTK and then compared to the solvent treated control sample (lane 1) of each experiment.
Supplementary Table S6. Genes with increased expression upon lestaurtinib treatment of KM-H2 as determined by GeneChip Human 1.0 ST Arrays. Absolute fold increase P-value Gene symbol Absolute fold increase P-value Gene symbol 3,7144306 0,00348652 TCP11L2 2,2225406 0,00386219 NCF2 3,5849998 0,0100037 GPR155 2,222141 2,86E-04 SOS2 3,2465725 0,00293521 DDIT4 2,2200117 0,02134405 GPR15 2,87905 0,00939321 PIK3IP1 2,205323 0,02235409 CES3 2,8498006 0,02188519 EVI2B 2,2043905 0,00416766 IFIT2 2,846551 2,84E-04 SH3D19 2,196642 0,00438728 LPXN 2,7736945 0,00801622 TNFSF8 2,1861305 0,00593732 DEPDC6 2,7407727 0,01262093 ABAT 2,1823678 0,03391139 C3AR1 2,7114697 0,00435157 ANTXR2 2,1773226 0,02661158 TRAF3IP3 2,6783195 0,00177221 KLHL24 2,1754122 0,01069166 SLFN5 2,5945666 0,01887752 CECR1 2,1749175 0,00685925 RGS1 2,592463 0,00284499 F2RL2 2,1728086 0,03651816 HIST1H2BK 2,584594 0,01087518 CAP2 2,150933 0,01360711 C3orf59 2,5774698 0,04359526 TMEM156 2,1435003 0,02465708 TMEM229B C14orf83 2,5356073 0,01535325 LIPH 2,1344452 0,01300903 SLC2A3 2,4619684 0,02230927 ABHD4 2,1240065 1,48E-04 CYTH1 2,439458 0,01499992 YPEL3 2,1182854 0,0408776 FBXO32 2,4356816 0,00222822 NEDD9 2,1041303 0,01191602 TLR6 2,3882196 4,60E-04 C4orf34 2,1009257 0,01585103 ACSF2 2,382293 3,62E-04 ACTA2 2,099514 0,01296231 STAT4 2,3743653 0,01366849 YPEL2 2,0870552 0,02936846 RARRES3 2,3728895 0,00910958 PIM2 2,0628216 0,00127561 TRGV3 TARP 2,3451083 0,00756518 GOLGA5 2,058902 0,03807717 FCGBP 2,340068 0,02344283 ENPP2 2,057207 5,15E-04 PDGFRA 2,337422 0,03027985 CCDC92 2,0566127 0,02318895 CD82 2,3373134 0,00103729 TFEC 2,0433033 0,02360682 CD48 2,3367746 0,00499932 TFPI 2,0211124 0,00244304 ENTPD1 2,3161943 0,01520138 VWA5A 2,018783 0,011257 DAP 2,2809763 0,00849459 BTG2 2,0177069 0,00609812 LOC285456 2,270525 9,94E-04 TNFSF10 2,0074186 0,0161396 CASP9 2,2384374 0,008908 MVP 2,004866 0,00392486 LY75 CD302 2,2302606 0,01186865 ST6GAL1 Absolute fold increases of genes in KM-H2 cells treated with 1 µm lestaurtinib for 24 h compared to a solvent treated control calculated from duplicate DNA microarray analyses by pairwise comparison (p < 0.05). Listed are 63 genes with fold changes > 2.
Supplementary Table S7. Genes with decreased expression upon lestaurtinib treatment of KM-H2 as determined by GeneChip Human 1.0 ST Arrays. Absolute fold P-value Gene symbol Absolute fold P-value Gene symbol decrease decrease 4,947711 0,00208741 HIST1H1B 2,4264805 0,0023599 KIF2C 3,8394911 0,00295397 KIF20A CDC23 2,4155557 4,69E-04 LOC100289612 3,8177176 0,00508658 PBK 2,414122 1,35E-04 KIFC1 3,5508034 0,01487117 HIST1H2AB 2,4108148 0,00516767 CKS1B LOC652 904 3,4745493 0,00134501 FOXM1 2,4068227 6,84E-04 CDC20 3,438392 0,00248822 MYBL2 2,4057395 0,02571027 H2AFX 3,3893778 0,00409837 CDK1 CDC2 2,3964353 0,00620524 HIST1H1A 3,2671223 2,52E-05 ANLN 2,3962715 0,00333325 E2F8 3,230833 0,00115335 NCAPG LCORL 2,385292 5,61E-04 CENPN C16orf61 3,2290049 0,00354436 NUF2 2,3828478 0,01000463 CENPA 3,2065177 0,0012671 CDCA2 2,3705285 0,00223835 UBE2C 3,162032 0,00110152 ESPL1 2,370174 9,77E-04 GMNN 3,152092 0,01902398 CCNA2 2,3699613 0,00515422 CENPK 3,148559 0,00272006 ASPM 2,3678763 0,00501668 HIST2H2AB 3,1330376 0,00412725 UHRF1 2,3604467 0,00566042 NUSAP1 3,073146 3,00E-04 CKAP2L 2,3531902 0,00782973 CSNK1G1 LOC100128564 3,072781 0,00534115 FAM72D FAM7 2,3502202 0,00134351 SKA1 2A FAM72B FA M72C 3,0724652 0,00317224 SPC25 2,34409 0,00320615 ERCC6L 3,0722387 0,00499735 FAM72D FAM7 2,3362653 0,00130279 WEE1 2A FAM72B FA M72C 3,0617206 0,00630812 KIF23 2,3346677 0,00455984 CCNF 3,051399 0,00581828 FAM72D FAM7 2,3339946 0,00101276 PEG10 2A FAM72B FA M72C 3,0510345 0,03987323 RRM2 2,3327193 3,83E-05 PRC1 3,0322857 9,58E-05 SPAG5 SGK494 2,3255339 5,20E-04 Transcripts Cluster Id: 7917752 3,0219114 0,01435495 PLK1 2,32453 0,00135952 AURKA 3,021459 0,00884133 HIST1H2BM 2,3186605 0,02734613 DHFR 2,9929626 0,00527378 FAM72D FAM7 2,316185 0,00278437 TK1 2A FAM72B FA M72C 2,9918034 0,00188516 CASC5 2,3142977 0,02152218 CENPW C6orf1 73 2,989064 0,00609754 KIF15 2,3083882 8,70E-04 SGOL2 2,9857385 0,00352462 AURKB 2,296972 0,00562388 UBE2S 2,9592016 0,00410746 DLGAP5 2,2945764 0,0059195 CDCA7L 2,92987 0,0040866 CENPE 2,2909012 0,0021914 FAM54A 2,8793344 0,00254328 ARHGAP11A 2,2902718 0,01021785 E2F7 2,863497 0,00152491 KIF4A 2,2826273 0,01448462 HIST1H4B 2,8629913 0,00383366 DHRS2 2,2723188 0,00343715 KIAA0101 CSN K1G1 2,8487034 7,57E-04 MCM10 2,2679121 0,00646454 MND1 2,8485816 0,00285708 NDC80 2,2483878 0,00834344 Transcripts Cluster Id: 8017096 2,84506 0,00264041 BUB1B PAK6 2,2470655 0,00101207 CIT 2,8293476 0,00149751 CDCA3 2,2368991 0,00458752 ZWINT 2,8169081 0,00795909 KIF18B 2,2344148 0,01304052 STIL 2,8106189 5,38E-04 ESCO2 2,233499 0,02454139 Transcripts Cluster Id: 7998927 2,809158 0,01364325 FAM111B 2,2200105 0,02229173 DHFR 2,8087556 0,01179081 DLEU2 2,2179043 0,02420177 XRCC2 2,8077152 0,00291433 CDCA8 2,1937153 0,01499939 PIGW 2,7992811 0,00595714 PLK4 2,1870885 0,00387937 CDC45 CDC45L 2,79299 0,00836114 GINS1 2,1868517 3,78E-04 RAD51 2,748775 0,00286333 KIFC1 2,1845288 0,00563364 MAD2L1 2,7355359 0,00350894 KIF14 2,1832361 0,01602458 NCAPH 2,7329178 0,00255445 HJURP 2,1805315 0,02078021 KIF20B 2,7280521 0,00216744 BUB1 2,1791623 0,01155007 CCNB2 2,725407 0,01547079 EXO1 2,1717613 0,01036931 TUBB2C 2,7154279 0,01121111 CDKN3 2,1630516 0,00954951 WEE1 2,709365 0,0026173 CDCA5 2,1630244 0,00347526 KIF18A
2,7050993 0,00324747 KIAA1524 2,158932 0,00313837 RFC3 2,694743 0,00364639 HIST2H3A HIST 2,1537757 0,00223091 DSCC1 2H3C HIST2H3 D 2,6947055 0,00364463 HIST2H3A HIST 2,1513839 0,00109221 RAD54L 2H3C HIST2H3 D 2,664744 4,61E-04 HIST1H2BI 2,1374402 5,57E-04 SPC24 2,6566284 0,00487014 SLC29A1 2,1318471 0,00255233 TMPO 2,6552584 1,08E-04 KIF18B 2,1210396 0,00729813 MRTO4 2,644857 8,61E-04 BIRC5 2,10888 0,0335351 FAM83D 2,6231754 8,54E-04 HMMR 2,1009142 0,0060417 TRAIP 2,5903828 0,0087509 SKA3 MRP63 2,0987954 7,93E-04 PRR11 2,5868797 0,00215522 RAD51AP1 2,0986414 0,00142361 C11orf82 2,5837846 0,01847473 DEPDC1 2,097215 2,06E-04 C3orf26 2,5701375 0,00166875 CENPF 2,09378 0,00328426 PKMYT1 2,5636146 9,16E-04 OIP5 2,0904844 0,0140051 Transcripts Cluster Id: 8175153 2,5622718 0,00468586 TPX2 2,0892031 0,03653432 PTTG1 2,561678 0,00288121 HIST2H3D HIST 2,0886962 0,00167859 TROAP 2H3A HIST2H3 C 2,5531638 0,0022266 CCNB1 2,082 0,02205829 GTSE1 2,5327036 0,00975182 TTK 2,0802782 0,0014747 HIST1H3D HIST 1H2AD 2,5291266 0,00428737 HIST1H4A 2,0694225 0,00249928 RACGAP1 2,520256 0,00272991 NEK2 2,061983 0,01277862 GSG2 2,5140715 0,00300028 HMGB2 2,0549028 0,00405064 CKS1B 2,5133893 0,01833098 CENPI 2,0538714 0,00696775 NEIL3 2,5032248 9,40E-04 TACC3 2,0534806 0,02144387 CENPH 2,5029547 0,00122392 CEP55 2,0491276 0,00300438 PRIM1 2,4982784 0,00277664 HIST2H3D HIST 2,0472429 0,00650959 ECT2 2H3A HIST2H3 C 2,4974785 0,00895417 C15orf42 2,0451565 0,04068505 HIST1H2BF 2,4936051 0,00676799 HIST1H2AK HIS 2,044454 0,0070631 HIST1H3G T1H2BN 2,472505 0,00362508 CKS1B LOC652 904 2,034462 7,47E-04 PCNA PCNAAS 2,4644399 0,00654321 CDC6 2,0326219 0,00634198 TRIP13 BRD9 2,4642212 0,00784964 UBE2T 2,0277228 0,01223802 NET1 2,4586377 0,0077142 TOP2A 2,0183227 8,71E-05 ARHGAP19 2,4458778 0,01717361 ORC1L 2,0165565 0,01874431 MMP12 2,4428658 0,00367476 HIST1H2AH 2,009581 0,00758611 ANP32E 2,4425745 0,00885502 DIAPH3 2,0092547 0,00579354 FARSB 2,4423397 0,00603805 KIF11 2,00754 0,00645785 DTL 2,4411573 0,00331908 MKI67 2,007142 0,00959901 CCDC99 2,4405727 2,04E-04 MELK 2,0061274 0,0090612 BRCA2 2,4285002 0,00419673 SGOL1 2,0044022 0,00330418 ATAD2 2,4281886 0,00833771 UBE2S 2,0037804 1,83E-04 DNA2 Absolute fold decreases of genes in KM-H2 cells treated with 1 µm lestaurtinib for 24 h compared to a solvent treated control calculated from duplicate DNA microarray analyses by pairwise comparison (p < 0.05). Listed are 180 genes with fold changes > 2.
Supplementary Table S8. Gene ontology annotation of differentially expressed genes in lestaurtinib treated KM-H2 cells. Number of annotated genes Gene ontology annotation Description (biological process) Decreased expression 56 GO:0000278 mitotic cell cycle 52 GO:0051301 cell division 47 GO:0007049 cell cycle 39 GO:0007067 mitosis 32 GO:0000087 M phase of mitotic cell cycle 29 GO:0000236 mitotic prometaphase 20 GO:0006334 nucleosome assembly 18 GO:0008283 cell proliferation 16 GO:0007059 chromosome segregation 15 GO:0006260 DNA replication Increased Expression 8 GO:0007165 signal transduction 7 GO:0006955 immune response 5 GO:0006915 apoptosis 4 GO:0007186 G-protein coupled receptor protein signaling pathway 4 GO:0016042 lipid catabolic process 3 GO:0009615 response to virus 3 GO:0043123 positive regulation of IκB kinase/nf-κb cascade 3 GO:0006919 activation of caspase activity 3 GO:0008283 cell proliferation Gene ontology (GO) analyses of genes with decreased or increased expression (fold changes > 2) after 24 h treatment of KM-H2 cells with 1 µm lestaurtinib compared to a solvent treated control determined by GeneChip Human 1.0 ST Array. Of 180 downregulated genes 116 were associated with the ten most strongly represented GO annotations shown here. Of 63 upregulated genes 25 are represented in the nine GO annotations listed.