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Supplementary Information

Treatment Specific Changes in Gene Expression Discriminate in vivo Drug Response in Human Leukemia Cells

D. Common Gene Expression Changes After ALL Treatments

Figure 6: Commonly regulated genes after all four initial treatments

Displayed are the median and the inter-quantile range of the 17 genes that exhibited higher expression after treatment (red) or lower expression after treatment (green), in at least 70% of patients, across all four treatments (Wilcoxon’s rank sum test P<0.01).

Figure 6: Commonly regulated genes after all four initial treatments

Table 4 (a) and (b): Changes in gene expression after single agent chemotherapy versus the same agents in combination

(a) Of the 97 gene probe sets that changed by more than 50% in at least 70% of patients after treatment with HDMTX alone, 38 increased and 59 decreased. As shown in Table D (1) below, only 21.1% of genes (8/38) that were up-regulated after HDMTX alone were also up-regulated when HDMTX was given with MP. Further, only 11.9% of genes (7/59) were down-regulated after HDMTX alone and also down-regulated after HDMTX when given with MP. The genes that concordantly changed after HDMTX alone and after HDMTX+MP are listed below. Negative numbers indicate a decrease and positive an increase in expression.

(a)probe set ID accession number gene name median FC
HDMTX
median FC
HDMTX+MP
36161_at M34175 adaptor-related protein complex 2, beta 1 subunit 5.7 3.6
37277_at U80017 baculoviral IAP repeat-containing 1 2.6 2.4
38819_at U33635 PTK7 protein tyrosine kinase 7 2.5 2.4
36651_at X15525 acid phosphatase 2, lysosomal 2.5 1.9
40123_at D87435 golgi-specific brefeldin A resistance factor 1 2.2 2.0
34279_at AL050141 hypothetical protein FLJ20719 2.0 2.5
32125_at AA928996 Tho2 1.9 3.0
38464_at X87237 glucosidase I 1.9 3.2
36432_at AL079298 methylcrotonoyl-Coenzyme A carboxylase 2 (beta) -1.8 -2.0
35074_at AF004715 jerky homolog-like (mouse) -1.9 -2.5
36246_at Z35309 adenylate cyclase 8 (brain) -2.2 -2.8
32413_at M13934   -2.8 -2.4
32583_at J04111 v-jun sarcoma virus 17 oncogene homolog (avian) -3.5 -2.3
725_i_at J03071   -4.0 -5.5
1915_s_at V01512   -5.7 -2.4

(b) Of the 197 probe sets that changed by more than 50% in at least 70% of patients after treatment with MP alone 40 increased and 157 decreased. As shown in Table 4 (b) below, only 17.5% of genes (7/40) that were up-regulated after MP alone were also up-regulated when MP was given with HDMTX and only 11.4% of genes (18/157) that were down-regulated after MP alone were also down-regulated after MP when given with HDMTX. The genes that concordantly changed after MP alone and after HDMTX+MP are listed below. Negative numbers indicate a decrease and positive an increase in expression.

(b) probe set ID accession number gene name median FC
MP
median FC
HDMTX+MP
36161_at M34175 adaptor-related protein complex 2, beta 1 subunit 4.0 3.6
32125_at AA928996 Tho2 2.5 3.0
35436_at L06147 golgi autoantigen, golgin subfamily a, 2 2.1 2.5
34836_at U18420 RAB5C, member RAS oncogene family 2.0 1.9
36822_at U51334 TAF15 RNA polymerase II, TATA box binding protein 2.0 1.9
37277_at U80017 baculoviral IAP repeat-containing 1 2.0 2.4
38915_at AB011135 KIAA0563 gene product 2.0 1.9
31652_at AB023217 KIAA1000 protein -1.9 -2.2
41117_s_at AB016243 solute carrier family 9 (sodium/hydrogen exchanger) -2.0 -1.7
38146_at AB011107 zinc finger protein 387 -2.1 -3.5
940_g_at D12625 neurofibromin 1  -2.1 -1.7
31785_f_at U92817 unnamed HERV-H protein -2.3 -2.5
34702_f_at M27826 chorionic somatomammotropin hormone 2 -2.3 -2.9
41303_r_at AI378632 Homo sapiens mRNA; cDNA DKFZp564P233 -2.5 -3.6
450_g_at U66469 cell growth regulatory with ring finger domain -2.5 -1.9
40590_at AA166687 cell division cycle 27 -2.8 -7.7
31529_at X99141 keratin, hair, basic, 3 -3.0 -2.1
39407_at M22488 bone morphogenetic protein 1 -3.0 -4.3
33047_at AI971169 ESTs, Highly similar to  BCL2-like 11 -3.2 -2.1
34704_r_at AA151971 chorionic somatomammotropin hormone 2 -3.5 -3.0
40387_at U80811 endothelial differentiation,G-protein-coupled receptor -3.5 -3.7
32583_at J04111 v-jun sarcoma virus 17 oncogene homolog (avian) -3.7 -2.3
39586_at AF097935 desmoglein 1 -4.0 -3.0
1915_s_at V01512   -4.9 -2.4
725_i_at J03071   -9.2 -5.5

Table 5 (a) and (b): Human leukemia cell lines differ from primary ALL cells.

(a) When the treatment with HDMTX alone (12 nM x 24 h plus 18 h drug-free media) was recapitulated (i.e., comparable level of cytotoxicity), with two human ALL cell lines in vitro (i.e., B-lineage Nalm6 [N.MTX] and T-lineage CEM [C.MTX]), we found very little overlap in the genes that changed after treatment in the cell lines compared to the primary leukemia cells in patients. Specifically, only seven out of the 97 genes (7.2%) that changed by more than 50% in at least 70% of patients after HDMTX also changed on average by more than 50% in the cell lines. Table 5 (a) lists the genes that concordantly change after HDMTX in cell lines and in patients, negative numbers indicate a decrease and positive an increase in expression.

(a) probe set ID accession number gene name median FC
N.MTX
median FC
C.MTX
median FC
HDMTX
32264_at L23134 granzyme M (lymphocyte met-ase 1) 1.9 1.6 2.9
36591_at X06956 tubulin, alpha 1 (testis specific) 1.9 1.2 1.9
U81800 solute carrier family 16 (monocarboxylic acid transporters), 1.5 1.7 3.6
2067_f_at L22475 BCL2-associated X protein 7.0 1.0 3.7
2001_g_at U26455 ataxia telangiectasia mutated 2.1 1.6 2.5
35692_at AL080235 Ras-induced senescence 1 -2.6 -1.0 -2.5
1916_s_at V01512 c-fos FBJ murine osteosarcoma viral oncogene homolog -1.4 -1.9 -11.3

(b) When the treatment with MP alone (10 µM x 24 h) was recapitulated with two human ALL cell lines in vitro (i.e., B-lineage Nalm6 [N.MP] and T-lineage CEM [C.MP]), we found very little overlap in the genes that changed after treatment in the cell lines compared to the primary leukemia cells in patients. Only 27 of the 197 genes (13.7%) changed in a consistent manner by more than 50% after MP treatment of cell lines and primary cells in vivo. Table 5 (b) lists the genes that concordantly changed after MP in cell lines and in patients as negative numbers indicate a decrease and positive an increase in expression.

(b) probe set ID accession number gene name median FC
N.MP
median FC
C.MP
median FC
MP
37881_at AF100907 growth differentiation factor 11 2.6 3.7 2.1
38547_at Y00796 integrin, alpha L (antigen CD11A (p180), lymphocyte function 4.9 -1.4 1.7
39286_at D64109 transducer of ERBB2, 2 1.9 3.0 3.0
40329_at AL031228 ring finger protein 1 1.3 3.2 1.7
41743_i_at AF061034 tumor necrosis factor alpha-inducible cellular protein 1.7 1.5 2.3
34335_at AI765533 ephrin-B2 1.6 1.5 1.7
34818_at X96381 ets variant gene 5 (ets-related molecule) 2.5 1.5 2.0
40951_at AL049250   1.7 1.7 1.9
292_s_at L29219 CDC-like kinase 1 1.1 2.5 1.7
31777_at AF006464 muscle, skeletal, receptor tyrosine kinase 1.2 -5.7 -2.6
33069_f_at U06641 UDP glycosyltransferase 2 family, polypeptide B15 -2.5 1.1 -1.7
34068_f_at X86174 synovial sarcoma, X breakpoint 1 -1.1 -2.5 -2.8
35081_at D14838 fibroblast growth factor 9 (glia-activating factor) -1.0 -4.9 -2.3
35109_at AB018299 KIAA0756 protein -5.3 -1.0 -2.6
37871_at X68830 islet amyloid polypeptide -3.0 -0.9 -2.0
40322_at D12763 interleukin 1 receptor-like 1 -1.1 -4.6 -2.1
40387_at U80811 endothelial differentiation, lysophosphatidic acid  -4.3 1.4 -3.5
32083_at AF027826 transmembrane 7 superfamily member 1 (upregulated in kidney) -1.0 -10.6 -3.2
35178_at W27944 Wnt inhibitory factor-1 -6.5 1.1 -5.3
39407_at M22488 bone morphogenetic protein 1 -1.2 -3.7 -3.0
32834_r_at AF013591 sudD (suppressor of bimD6, Aspergillus nidulans) homolog -3.7 -1.1 -3.0
39448_r_at W27095 B7 protein -1.9 -1.3 -1.6
41244_f_at X80910 protein phosphatase 1, catalytic subunit, beta isoform -2.6 -1.7 -2.1
32531_at X52947 gap junction protein, alpha 1, 43kD (connexin 43) -1.2 -5.7 -3.2
32583_at J04111 v-jun sarcoma virus 17 oncogene homolog (avian) 2.0 -16.0 -3.7
1152_i_at J00117 chorionic gonadotropin, beta polypeptide -2.8 -2.3 -6.1
618_at M26167 platelet factor 4 variant 1 -2.1 -4.3 -4.0