1. 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

2. Figure 2 ABC 0 1 2 3 Luc  HCT116-s p38 Sh RNA IC50 (nM) 0 1 2 3 EV   HCT116-s p38 CA isoforms IC50 (nM) F HCT116-SN6 0 5 10 15 20 25 Luc   p38 Sh RNA G 0 5 10 15 20 25 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) 0 1 2 3 EV  +  p38 CA isoforms HCT116-SN6 IC50 (nM) 0 4 8 12 16 EV  p38 CA isoforms H D

3. Figure 3 A E 0 1 2 3 4 5 6 7 SN38SN38+SB SW480 IC50 (nM) 0 2 4 6 8 10 12 SN38SN38+SB HT29 B C 0 1 2 3 4 IC50 (µM) 5-FU5-FU+SB HCT116-s 0 200 400 600 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) 0 2 4 6 8 10 12 EV p38  SW480 IC50 (nM) p38 CA isoforms HT29 0 2 4 6 8 10 12 14 EV p38  p38 CA isoforms D HCT116-SN6 SN38 IC50 (nm) SN38SBSN38 +SB SB IC50 (µm) 0 2 4 6 8 10 0 5 15 20 25   HCT116-s SN38SBSN38 +SB SN38 IC50 (nM) 0,0 0,5 1,0 1,5 2,0 0 5 10 15 20 SB IC50 (µM)  

4. Figure 4 Irinotecan 40 mg/ml IP AB SB202190  p< 0.05,  p< 0.01,  p<0.001 C % mice with tumor size doubled

5. Figure 5 AB C Response nuclear QS Responder Non Responder Total <1478513 >147178 Total91221 1 2 3 4 5 6

6. 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.

7. A C Clone 1Clone 2 MKK6 CA EV SW480 FLAG pATF2 WB KA 0 1 2 3 4 5 6 7 8 EVMKK6 CA Clone 1 MKK6 CA Clone 2 IC50 (nM) SW480 IC50 (nM) HCT116 0 1 2 3 4 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

8. A C B  -actin shLuc Shp38α pMSCV pMSCVp38β shLuc Shp38α pMSCV pMSCVp38β Topo I HCT116-sHCT116-SN6 1.3 1.2 1.6 2.3 1.3 1.5 1.7 2.5 Sh Luc Shp38  HCT116-SN6HCT116-s Sh Luc Shp38  NT Sn38 1µM 0 20 40 60 80 100 120 140 ShLuc Shp38  HCT116-SN6HCT116-s ShLuc Shp38  ICE arbitrary units SN38 treated samples HCT116-SN6 pMSCV p38  NT Sn38 1µM HCT116-SN6 pMSCV p38  0 20 40 60 80 100 120 140 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.

9. AB 1 2 3 4 5 6 7 8 pMSCV p38 beta  -actin p38  D Days Tumor size (mm 3 ) 0 100 200 300 400 500 600 051015 20 25 HCT116-SN6 pMSCV HCT116-SN6 p38  Irinotecan 40 mg/ml IP Days Tumor size (mm 3 ) 0 100 200 300 400 500 600 0510152025 HCT116-SN6 Sh Luc HCT116-SN6 Sh p38  Irinotecan 40 mg/ml IP C 0 100 200 300 400 500 600 051015 2025 HCT116-SN6 pMSCV HCT116-SN6 p38  CA HCT116-SN6 Sh Luc HCT116-SN6 Sh p38  Days Tumor size (mm 3 ) 1 2 3 4 5 6 7 8 Sh Luc Sh p38   -actin p38  E HCT116-SN6 0 10 20 30 40 50 60 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.