Figure 1 A -SN50-C8-S-A2-G7 B C HCT116-sHCT116-SN6 NTSN38NTSN38 HCT116-s pp38 tubulin NT 0.1µM 1µM 5µM 10µM pp38 tubulin HCT116-SN6 [SN38] 24H pp38 NT 1h 4h 7h 16h 24h 48h tubulin pp38 tubulin HCT116-s HCT116-SN6 SN38 1µM HCT116-SN6

Figure 2 ABC Luc  HCT116-s p38 Sh RNA IC50 (nM) EV   HCT116-s p38 CA isoforms IC50 (nM) F HCT116-SN Luc   p38 Sh RNA G EV  p38 CA isoforms HCT116-SN6 IC50 (nM) p38δ p38α p38  p38  Sh Luc Sh p38 EVp38 CA HCT116-s  -Actin E p38δ p38α p38  p38  Sh Luc Sh p38 EVp38 CA HCT116-SN6  -Actin  p< 0.05,  p< 0.01,  p<0.001 HCT116-s IC50 (nM) EV  +  p38 CA isoforms HCT116-SN6 IC50 (nM) EV  p38 CA isoforms H D

Figure 3 A E SN38SN38+SB SW480 IC50 (nM) SN38SN38+SB HT29 B C IC50 (µM) 5-FU5-FU+SB HCT116-s IC50 (nM) OxaliOxali+SB HCT116-s pp38  -Actin p38 NT5-FUOxali HCT116-sHCT116-SN6 NTSN38 + SB NTSN38 + SB pATF2 F  p< 0.05,  p< 0.01,  p<0.001 IC50 (nM) EV p38  SW480 IC50 (nM) p38 CA isoforms HT EV p38  p38 CA isoforms D HCT116-SN6 SN38 IC50 (nm) SN38SBSN38 +SB SB IC50 (µm)   HCT116-s SN38SBSN38 +SB SN38 IC50 (nM) 0,0 0,5 1,0 1,5 2, SB IC50 (µM)  

Figure 4 Irinotecan 40 mg/ml IP AB SB  p< 0.05,  p< 0.01,  p<0.001 C % mice with tumor size doubled

Figure 5 AB C Response nuclear QS Responder Non Responder Total < > Total

SW48-s SW48-SN1 SW48-SN2 SW48-SN3SW48-SN4 pp38 tubulin IC50 (nM) Resistance Factor SW48-s6,1± 0,81 SW48-SN1377,5 ± 3762 SW48-SN2262,0 ± 2443 SW48-SN3121,0 ± 1720 SW48-SN4279,3 ± 5346 AB Supplemental Figure 1: p38 is activated by phosphorylation in SN38-resistant SW48 cells. A: Drug sensitivity of the SW48 clones: IC50 values were determined using the SRB assay; the resistance factor was determined by dividing the IC50 value of each resistant clone by that of the sensitive clone SW48-s. Data represent the mean ±SD of at least 3 independent experiments. B: Western blot analysis of p38 phosphorylation in SW48-s cells and in the SN38 resistant clones SW48-SN1, SW48- SN2, SW48-SN3, SW48-SN4.

A C Clone 1Clone 2 MKK6 CA EV SW480 FLAG pATF2 WB KA EVMKK6 CA Clone 1 MKK6 CA Clone 2 IC50 (nM) SW480 IC50 (nM) HCT EV MKK6 CA FLAG MKK6 CA EV WB HCT116-s pp38 WB tubulin p38 WB Supplemental Figure 2: Analysis of the involvement of MKK6 CA in SN38 resistance. A: Western blot analysis of HCT116 cells that express constitutively active (CA) MKK6 (or EV, as a control). MKK6 overexpression was detected with anti-FLAG antibody. p38 expression and activation were detected with anti-p38 and anti-phospho-p38 antibodies respectively. Equal loading is shown by tubulin expression. B: SRB assay on HCT116 expressing MKK6 CA or EV as control, and treated with SN38. C: Western blot analysis of 2 clones of SW480 cells that stably express constitutively active (CA) MKK6 (or EV, as a control). MKK6 overexpression was detected with anti-FLAG antibody. p38 activation was detected using a kinase assay to test p38a activity in the 2 clones stably expressing MKK6 CA or Empty Vector as a control. D: SRB assay on SW480 expressing MKK6 CA or EV as control, and treated with SN38. B D

A C B  -actin shLuc Shp38α pMSCV pMSCVp38β shLuc Shp38α pMSCV pMSCVp38β Topo I HCT116-sHCT116-SN Sh Luc Shp38  HCT116-SN6HCT116-s Sh Luc Shp38  NT Sn38 1µM ShLuc Shp38  HCT116-SN6HCT116-s ShLuc Shp38  ICE arbitrary units SN38 treated samples HCT116-SN6 pMSCV p38  NT Sn38 1µM HCT116-SN6 pMSCV p38  ICE arbitrary units SN38 treated samples Supplemental Figure 3: Analysis of Topoisomerase I expression and activity. A: Western blot analysis of Topoisomerase I (TopoI) in HCT116-s-ShLuc, HCT116-s-Shp38a, HCT116-s-pMSCV and HCT116-s-CAp38b cells and in HCT116-SN6-ShLuc, HCT116- SN6-Shp38a, HCT116-SN6-pMSCV and HCT116-SN6-CAp38b cells. Equal loading is shown by b-Actin expression. Numbers underneath the b-actin panel are the quantification data for total TopoI level obtained from western blot analysis after normalization to β-actin. B: Quantification of SN38-induced TopoI-DNA complexes using the ICE bioassay and nuclear extracts from HCT-116-s- ShLuc and -Shp38a cells and HCT116-SN6-ShLuc and -Shp38a cells. The relative intensity of the immune complexes in SN38- treated cells was normalized to that of untreated cells. C: Quantification of SN38-induced TopoI-DNA complexes using the ICE assay and nuclear extracts from HCT116-SN6-pMSCV and -CAp38b cells. The relative intensity of the immune complexes in SN38-treated cells was normalized to that of untreated cells.

AB pMSCV p38 beta  -actin p38  D Days Tumor size (mm 3 ) HCT116-SN6 pMSCV HCT116-SN6 p38  Irinotecan 40 mg/ml IP Days Tumor size (mm 3 ) HCT116-SN6 Sh Luc HCT116-SN6 Sh p38  Irinotecan 40 mg/ml IP C HCT116-SN6 pMSCV HCT116-SN6 p38  CA HCT116-SN6 Sh Luc HCT116-SN6 Sh p38  Days Tumor size (mm 3 ) Sh Luc Sh p38   -actin p38  E HCT116-SN Sh Luc Sh p38 alpha p38  /  -actin Arbitrary units Supplemental Figure 4: The differential expression of the four p38 isoforms influences the response to irinotecan. A: Tumor growth kinetics in mice xenografted with HCT116-SN6- pMSCV or HCT116-SN6-p38 , HCT116-SN6-ShLuc or HCT116-SN6- Shp38  cells before irinotecan treatment. B: Tumor growth kinetics in mice xenografted with HCT116-SN6-ShLuc or HCT116- SN6-Shp38  cells and treated with irinotecan. C: Tumor growth kinetics in mice xenografted with HCT116-SN6-pMSCV or HCT116-SN6-p38  cells and treated with irinotecan. D: Western blot analysis of p38  expression in HCT116-SN6-pMSCV and HCT116-SN6-CA38  xenografts. Equal loading is shown using  -Actin. E: Western blot analysis of p38  expression in HCT116-SN6-ShLuc or HCT116-SN6-Shp38  xenografts. Equal loading is shown using  -Actin. Histogram shows the quantification data for p38  level obtained from western blot analysis after normalization to β-actin.