A UP RESPONSE TO HYPOXIA DOWN

NES 2.5 FDR 0% NES -2.1 FDR 0%

Higher expression in resistant Higher expression in sensitive B ATF4 CEBPB CEBPG

NES 3.2 FDR 0% NES 2.2 FDR 0% NES 2.0 FDR 0%

ATF3 JUN JUNB

NES 2.0 FDR 0% NES 1.4 FDR 0% NES 1.1 FDR 3%

Higher expression in resistant Higher expression in sensitive

Figure S1. set enrichment analysis in daunorubicin-resistant versus sensitive K562 cells. Related to Figure 2.

(A) GSEA plots show enrichment of a hypoxia gene signature (12) in resistant versus sensitive K562 cells. (B) GSEA plots show enrichment of the top 500 target for the indicated transcription factors in resistant versus sensitive K562 cells. The GSEA plot for ATF4 is the same as shown in Figure 2E and is included for reference. A ABCB4 ABCB1 RUNDC3B

80th centile Median 20th centile 1 0.1 0.01 0.001 100 kb hg38 Chr7:87,375,000-87,828,125

B 20 kb hg38 ABCB1 C1 E3 C2 P C3 K562_R1

K562_R2

K562_R3

H3K27ac Liver

Adrenal

CD34+ HSPCs 1 CD34+ HSPCs H3K4me

K562

DHS K562

CTCF

CTCF

CTCF

Transcription factor Transcription SMC3 80th centile Median 20th centile

4Cseq 1 0.1 0.01 0.001

Figure S2. Structure of the ABCB1 locus. Related to Figure 3.

(A) Contact profile for the region containing ABCB1 (Chr7: 87,375,000-87,828,125) generated from 4C sequencing of K562_R1 using a viewpoint centred on the ABCB1 promoter. (B) ChIPseq tracks for H3K27ac, H3K4Me1, CTCF and SMC3 in K562 cells (ENCODE); and DNAse-seq (ENCODE). Regions of contact that do not contain an active enhancer in K562_R1-3 are highlighted in red (C1-3). H3K27ac ChIPseq tracks and 4Cseq contact profile from K562_R1-3 are included for reference. A B Chr7:87,503,000-87,513,000 Chr7:87,624,500-87,626,000 100 100 5 kb hg38 1 kb hg38 E1 E2 E4 100 100 E1_5 E2_1 E2_2 E2_3 E2_4 E1_4 E2_5 E1_1 E1_2 E1_3 E4_2 E4_1 * * * C D Chr7:87,597,000- Chr7:87,570,000-87,577,000 87,602,000 5 kb hg38 2 kb hg38 10 10 E3 Promoter 10 10 P_5 P_1 P_2 P_3 P_4 P_6 E3_5 E3_9 E3_7 E3_8 E3_2 E3_4 E3_1 E3_3 E3_6 E3_11 E3_11 E3_13 E3_12 E3_10 Calcein * Calcein * E F E1 E2 E3 E4 100 1 1 CalceinCalcein MFI 5 CalceinCalcein MFI 1 1 10 ABCB1UIC2 MFI ABCB1UIC2 MFI 1 Fold change Fold change Foldchange Foldchange UIC2 UIC2 0.1 Fold change Fold change P_1 P_2 P_3 P_4 P_6 0.01 P_5 0.5 0.1 0.1 * E3_5 E2_5 E1_5 E3_11 E3_11 E3_12 E3_13 E3_10 E4_1 E3_6 E3_7 E3_8 E3_9 E3_4 E3_1 E3_2 E3_3 E2_1 E2_2 E2_3 E2_4 E1_3 E1_4 E1_1 E1_2 E4_2 0.1 0.1 * * * * Figure S3. Screening of sgRNAs for promoter and enhancer silencing. Related to Figure 3.

(A-D) ChIPseq tracks for H3K27ac in resistant K562 show the position of the sgRNA sequences designed to target the promoter (D) and each enhancer (A-C). (E) Bar chart shows the change in ABCB1 expression and calcein AM retention following dual infection of K562_R3 with pHR-SFFV-dCas9-BFP-KRAB and pLKO5.sgRNA.EFS.tRFP657 containing the indicated promoter-targeting sgRNA. (F) As for (E), but shows data

0.01 P_1 P_2 0.01 P_3 P_1 P_4 P_2 P_5 P_3 P_6 forP_4 each enhancer-targetingP_5 sgRNA.P_6 *indicates the sgRNA chosen for the promoter and each enhancer.

0.01 P_1 P_2 0.01 P_3 P_1 P_4 P_2 P_5 P_3 P_6 P_4 P_5 P_6 A 50 kb hg38

ABCB1

E1 E2 E3 P E4

2000

0 K562_R3 20

0 ATF4 20

0 ATF3 10

0 JUND 10

0 JUN 5

0 CEBPB 5

0 CEBPG 5

0 EP300 5

0 TAL1 5

0 GATA2

Figure S4. binding within ABCB1. Related to Figure 4.

(A) ChIPseq tracks for the indicated transcription factors in unmanipulated K562 (ENCODE) within the ABCB1 gene. H3K27ac ChIPseq tracks from K562_R3 are included for reference. The promoter (P) and enhancers (E1-4) are highlighted in blue. 18 18 18 16 18 16 18 16 18 14 16 14 16 14 16 12 14 12 14 12 14 EV EV EV 10 12 10 12 10 12 A B EV Vehicle C EV Vehicle D EV Vehicle 10 10 10 E4 Tosedostat E4 Tosedostat E4 Thapsigargin 8 8 NS 8 ** *** 1.2 4 2.0 ** 3 ** ** 6 8 6 8 p = 0.001 6 8 E4 ** E4 E4 ** 3 1.5 *** 1.0 2 ** 4 6 4 6 4 6 *** expression expression 2 1.0 ** **

18 18 JUN 18 2 4 2 4 0.8 ATF4 2 4 1 18 18 18 1 0.5 16 0 2 16 0 2 16 0 2 Relativeexpression Relativeluminescence Relative

0.6 Relative 0 0.0 0 14 16 0 VEH 14 16 100nM 0 VEH 500nM 14 16 100nM 0 S R1VEH R2500nM R3 100nM S R1 500nM R2 R3 ATF4 JUN ABCB1 DDIT3 DDIT4 12 14 VEH 12 14 100nM Na3VO4VEH 500nM 12 14 100nM VEH 500nM 100nM 500nM CEBPB VerapamilEV Tosedostat EV EV 10 12 10 12 10 12 E EV Low density F EV Vehicle G EV Vehicle High density Daunorubicin Daunorubicin 10 8 10 8 100 E4 10 8 *** 3 E4 2.0 E4 ** * * 6 8 6 8 E4 6 8 E4 1.5 E4 2 10 *** ** expression

*** expression 4 6 4 6 4 6 * 1.0 JUN 1 ATF4 1 2 4 2 4 2 4 0.5 Relativeexpression Relative 0 2 0 2 0.1 0 2 Relative 0 0.0 S R1 R2 R3 S R1 R2 R3 0 VEH 100nM 0 500nM VEH 100nM ATF40 VEH 500nM JUN 100nM 500nM ABCB1 DDIT3 DDIT4 VEH 100nM 500nM VEH 100nM CEBPBVEH 500nM 100nM 500nM Figure S5. Stress induces expression of ABCB1. Related to Figure 5.

(A) Mean±SEM relative luminescence from luciferase assay of ABCB1 ATPase activity following treatment with

Na3VO4 (an ABCB1 inhibitor), verapamil (a known ABCB1 substrate) or tosedostat (n=3). NS, not significant by one way ANOVA with Tukey post hoc test. (B-C) Mean±SEM expression of (B) ATF4 and (C) JUN by quantitative PCR relative to unmanipulated drug sensitive K562 cells following exposure to tosedostat 50µM for 48hrs (n=3). **p<0.01, ***p<0.001 by unpaired t-test. (D) Mean±SEM expression of the indicated genes by quantitative PCR relative to vehicle for unmanipulated drug sensitive K562 cells following exposure to thapsigargin 100nM for 48hr (n=3). **p<0.01, ***p<0.001 by unpaired t-test. (E) Mean±SEM expression of the indicated genes by quantitative PCR relative to drug sensitive K562 cells cultured at low density (<5x105 cells/ml) following 48hr of high density culture (>106 cells/ml, n=3). *p<0.05, **p<0.01, ***p<0.001 by unpaired t-test. (F-G) Mean±SEM expression of (F) ATF4 or (G) JUN by quantitative PCR relative to unmanipulated drug sensitive K562 cells following exposure to daunorubicin 100nM (S) or 500nM (R1-3) for 48hrs (n=3). *p<0.05, **p<0.01 by unpaired t-test. 100 100 100 100 100 100 10010 10010 Vehicle 10 Vehicle 10 Vehicle 100 10010 Vehicle 10nM 10nM 10010 100 Vehicle 101 Vehicle 10nM A Vehicle100nM 10nM BB893 1B 101 1000 BB889 2M 10 10nMVehicle 100 100 Vehicle100nM 100nM10nM 1 10nMVehicle 10nM 1000nM100nM 100101 10010 100 100nM100nM 1000nM 1 1000nM1000nM 10nM 10 Vehicle10nM 1 100nMVehicle 101 10 100nM 1000nM 100 1001 1 1000nM 0.1 Relativeexpression Vehicle Relativeexpression Vehicle100nM 0.1101 10 10nM100nM 1000nM10nM 100 100 0.1 1000nM 0.1 0.1 ABCB1VehicleATF4 DDIT3 DDIT4 CEBPB JUN ABCB1VehicleATF4 DDIT3 DDIT4 CEBPB JUN 0.1 10nM 10nM1000nM 101 101 ATF4 8 BB901 1B 1000 BB667 2M 100 100 DDIT3Vehicle100nM1000nM DDIT4 Vehicle100nM ATF4 0.1 ABCB1 Vehicle CEBPB Vehicle

10nM 0.1 DDIT3 DDIT4 10nM ABCB1 CEBPB ATF4 6 100nM10nM 100 100nM10nM 1001 1001 DDIT3100nM DDIT4 100nM 10 ATF4 0.110 ABCB1 1000nM CEBPB 1000nM DDIT3 DDIT4 1000nM 1000nM 0.1 ABCB1 CEBPB ATF4 4 Vehicle10nM 10 Vehicle10nM 1 1 DDIT3 DDIT4 10 10 ABCB1 100nM CEBPB 100nM 100 ATF4 100 2 1000nM 1 1000nM Relativeexpression DDIT3 DDIT4 Relativeexpression 1 ABCB1 1 CEBPB ATF4 Vehicle Vehicle 0.110 0.110 ABCB1 DDIT310nM100nM DDIT4 10nM100nM 100 ATF4 100 0 1000nM CEBPB 0.1 1000nM DDIT3 DDIT4 ABCB1VehicleATF4 DDIT3 DDIT4 CEBPB JUN ABCB1VehicleATF4 DDIT3 DDIT4 CEBPB JUN 0.1 ABCB1 0.1 CEBPB 10nM 10nM 101 101 B 6 BB54 2B 6 BB140 4B 100 100 Vehicle100nM1000nM Vehicle100nM1000nM ATF4 ATF4 Vehicle Vehicle

0.1 ABCB1 DDIT3 0.1DDIT4 ABCB1 DDIT35 10nM10nM DDIT4 5 10nM10nM 1 1 CEBPB 100nM CEBPB 100nM 100 100 4 100nM 4 100nM 10 ATF4 10 ATF4 1000nM1000nM 1000nM1000nM 0.1 ABCB1 DDIT3 0.1DDIT4 ABCB1 DDIT33 Vehicle10nM DDIT4 3 Vehicle10nM 1 1 CEBPB 100nM CEBPB 100nM 10 ATF4 10 ATF4 2 1000nM 2 1000nM Relativeexpression DDIT3 DDIT4 Relativeexpression DDIT3 DDIT4 ABCB1 ABCB1 1 Vehicle 1 Vehicle 0.11 0.11 CEBPB 10nM100nM CEBPB 10nM100nM 10 ATF4 10 ATF4 0 0 DDIT3 DDIT4 DDIT3ABCB11000nM ATF4DDIT4DDIT3 DDIT4 CEBPB JUN ABCB11000nMATF4 DDIT3 DDIT4 CEBPB JUN 0.1 ABCB1 0.1 ABCB1CEBPB Vehicle CEBPB Vehicle 10 10 10nM BB171 3B 10nM BB556 2B 1 1 6 1000nM 6 1000nM ATF4 ATF4 Vehicle100nMVehicle Vehicle100nMVehicle

DDIT35 10nM DDIT4 5 10nM 0.1 ABCB1 DDIT3 0.1DDIT4 ABCB1 10nM CEBPB 10nM 1 1 CEBPB 100nM 100nM 4 100nM 4 100nM ATF4 ATF4 1000nM 1000nM DDIT3 DDIT4 DDIT31000nM DDIT4 1000nM 0.1 ABCB1 0.1 ABCB1 3 10nM 3 10nM 1 1 CEBPB 100nM CEBPB 100nM ATF4 ATF4 2 1000nM 2 1000nM Relativeexpression DDIT3 DDIT4 Relativeexpression DDIT3 DDIT4 0.11 ABCB1 0.11 ABCB1CEBPB 1 100nM CEBPB 1 100nM ATF4 ATF4 0 0 DDIT3 DDIT4 DDIT3ABCB11000nM ATF4DDIT4DDIT3 DDIT4 CEBPB JUN ABCB11000nMATF4 DDIT3 DDIT4 CEBPB JUN 0.1 ABCB1 0.1 ABCB1CEBPB CEBPB Figure S6. Fresh primary AML blasts respond to acute daunorubicin exposure1000nM with ABCB1 induction. Related to ATF4 ATF4 1000nM DDIT3 DDIT4 Figure 6. DDIT3 DDIT4 0.1 ABCB1 0.1 ABCB1CEBPB CEBPB ATF4 ATF4(A-B) Mean±SEM relative expression of the indicated genes following exposure of fresh (A) or freeze-thawed (B) 0.1 DDIT3 0.1DDIT4 primary AMLDDIT3 blast cells toDDIT4 the indicated doses of daunorubicin for 18 hours (n=3). BB numbers indicate Biobank ABCB1 ABCB1CEBPB identifier. CEBPB ATF4 ATF4 DDIT3 DDIT4 DDIT3 DDIT4 ABCB1 ABCB1CEBPB CEBPB ATF4 ATF4 DDIT3 DDIT4 DDIT3 DDIT4 ABCB1 ABCB1CEBPB CEBPB 100 100

10 100 Vehicle BB946 2B 10 A B 18 *** 100 Vehicle p = 0.04 p = 0.016 16

Vehicle 100 14 1000nM 40 900 10nM*** 12 100 35 800 10 100 700 30 8 *** 600 10nM 1 25 ** expression 6 expression 500 100nM 20 ** **

Relativeexpression 4 10010 100 400 ATF4 15 DDIT3 2 1 300 Vehicle 10 10 0 100nM 10 100 200 ABCB11000nMATF4 DDIT3 DDIT4 CEBPB JUN Relative 5 Relative 100 Vehicle 0 0 Vehicle 10 7 BB953 2B *** ** 100 0 1 2 3 0 1 10nM2 3 Vehicle1000nM 10010 6

** 1000nMVehicle 0.1 Vehicle 5 10nM * 100C 10nM 100101 10100 4 * Fresh samples Vehicle100nM

0.1 10nMVehicle 3 10nM Vehicle 1 ABCG2 Ct values 10nM 100 ~30-33 (IQR) 100nM 2 100nM* 1 ATF410 10 1000nM100nM Relativeexpression 100expression DDIT3 DDIT4 1 ABCB1 CEBPB1000nM10nM 1 Vehicle 100101 ATF410 0 100nM 10nM 100ABCG2 1 DDIT3 DDIT4 100nM ABCB11000nMATF4 DDIT3 DDIT4 CEBPB JUN ABCB1 CEBPB1000nMVehicle Vehicle 10 D 10nM 101 1Relative 100 0.1 100nM Frozen samples 1000.1 1000nM Vehicle100nM 1000nMVehicle 10nMVehicle 0.1 10nM 10 10nM 0.110 1 100nM100nM 100 BB889 1B BB667 2M BB889 2M BB893 1B BB901 1B 10 1000nMVehicleexpression 1000nM1000nM 0.1ATF4 1 10 DDIT3 DDIT4E 1 10nM Vehicle10nM

0.1 ABCG2 1 ABCB1 100 Undetectable FreshCEBPB samples Vehicle100nM 100nM ATF410 DDIT3 DDIT4 10nMVehicle 0.1 1000nM ATF4ABCB1 ABCC1 Ct values ~23-24 (IQR) DDIT3 DDIT4 CEBPB100nM10nM Vehicle 0.1 1 Relative 1 ABCB1 0.110 CEBPB 100nM 10 1000nM100nM 0.01 10nM ATF4expression DDIT3 DDIT4 1000nM BB54 2B BB140 4B BB171 3B BB5561000nM 2B ATF4ABCB1 CEBPB10nM 1 ABCB1 DDIT3 0.1DDIT4 Vehicle

ABCC1 CEBPB 1 100nMF 10nM 101 100

Frozen samples 1000nM ATF4 ATF4 1000nM Vehicle100nM DDIT3 DDIT4 DDIT3 DDIT4 10nMVehicle 0.11 ABCB1 0.1Relative ABCB1CEBPB 100nM CEBPB 10nM 0.1 100nM 1 10 1000nM 1000nM100nM ATF4 expression DDIT3 DDIT4 1000nM 0.1 0.1 ABCB1 CEBPB 10nM 1 BB889 1B BB667 2M BB889 2M BB893 1B BB901 1B 1000nMABCC1 100nM ATF4 ATF4 1 DDIT3 DDIT4 DDIT3 DDIT4 1000nM 0.1 ABCB1 ABCB1CEBPB CEBPB

1 Relative 0.1 ATF4 100nM ATF4 DDIT3 DDIT40.1 1000nM 0.1 DDIT3 DDIT4ABCB1 BB54 2B BB140 4B BB171 3B BB556 2B ABCB1 0.1 CEBPB CEBPB ATF4 Figure S7. Fresh primary AML blasts respond to acute daunorubicin exposure with ABCB1 induction. Related to DDIT3 DDIT4 ATF4 1000nM ABCB1 Figure0.1 6. CEBPB DDIT3 DDIT4 ATF4 ABCB1 CEBPB DDIT3 (A) DDIT4Expression of ATF4 and ATF4DDIT3 relative to BB667 for all fresh and frozen primary AML samples. Fresh were ABCB1 compared with CEBPB frozen using an unpairedDDIT3 t-test. (B) Mean±SEMDDIT4 relative expression of the indicated genes following0.1 exposure ABCB1 of fresh primary AML blast cells (BB946 & BB953)CEBPB to 1000nM daunorubicin for 18 hours (n=3). *p<0.05, **p<0.01, ***p<0.001ATF4 by unpaired t-test. (C-D) Mean±SEM relative expression of ABCG2 following exposure of (C) fresh or (D) freeze-thawedDDIT3 AML blast cellsDDIT4 to the indicated doses of daunorubicin for 18 hours (n=3). (E-F)ABCB1 Mean±SEM relative expression of ABCC1 following exposureCEBPB of (E) fresh or (F) freeze-thawed AML blast cells to the indicated dosesATF4 of daunorubicin for 18 hours (n=3). BB numbers indicate Biobank identifier. IQR, interquartileABCB1 range. DDIT3 DDIT4 CEBPB chr16:15,932,215-16,144,747 A 100 kb hg38 ABCC1

75

0 K562_R1 75

0 K562_R2 75

0 K562_R3 5 H3K27ac 0 Liver 20

0 Adrenal 50 1 0 CD34+ HSPCs 20

H3K4me 0 CD34+ HSPCs 1.5

DHS 0 K562 15

0 EP300 15

0 JUND 15

0 CEBPB 10

0 CEBPG 10

Transcription factor Transcription 0 JUN 5

0 ATF4 5

0 ATF3

120 200 150

0 0 0 K562_R1 120 200 150

0 0 0 K562_R2 120 200 150

0 0 0 K562_R3 10 10 5 H3K27ac 0 0 0 Liver 20 15 10

0 0 0 Adrenal 50 50 130 1 0 0 0 CD34+ HSPCs 25 35 25

H3K4me 0 0 0 CD34+ HSPCs 5 10 2

DHS 0 0 0 K562 25 50 25

0 0 0 EP300 30 80 15

0 0 0 JUND 15 110 15

0 0 0 CEBPB 10 30 5

0 0 0 CEBPG 20 30 15

Transcription factor Transcription 0 0 0 JUN 15 15 10

0 0 0 ATF4 5 5 10

0 0 0 ATF3

Figure S8. Regulatory element landscape of ABCC1. Related to Figure 6.

(A) ChIPseq tracks for H3K27Ac, H3K4Me1 and DNAse-seq surrounding ABCC1 (chr16:15,932,215-16,144,747; hg38) in the indicated human cells and tissues, including CD34+ hematopoietic stem and progenitor cells (ENCODE) and K562_R1-3 (our data). Putative enhancers are highlighted in blue. ChIPseq tracks for the indicated transcription factors are for unmanipulated K562 cells from ENCODE (14). A 7000 ABCB1 6000 ATF4 16000 DDIT3

6000 5000 14000 5000 12000 4000 10000 4000 3000 8000 3000 2000 6000 2000 Signalintensity Signalintensity Signalintensity 4000 1000 1000 2000 0 0 0 ETP ETP ETP MLP BNK MLP BNK MLP BNK MPP MEP MPP MEP MPP MEP CMP CMP CMP GMP GMP GMP Pro-B Pro-B Pro-B HSC1 HSC2 HSC1 HSC2 HSC1 HSC2

Correlation B Gene p-value C (r) ATF4 DDIT3 ATF4 0.91 2.49 x 10-4 12000 5000 JUN 0.82 3.63 x 10-3 10000 4000 -3 JUNB 0.42 5.14 x 10 8000 3000 -4 XBP1 0.89 4.94 x 10 6000 2000 KLF6 NOT AVAILABLE signal intensity signal intensity 4000 -5 DDIT3 0.95 2.02 x 10 r = 0.91 1000 r = 0.95

ATF4 2000 p < 0.001 DDIT3 p < 0.001 CEBPB 0.78 7.77 x 10-3 0 0 KLF10 0.70 2.43 x 10-2 0 2000 4000 6000 0 2000 4000 6000

FOSB 0.81 4.32 x 10-3 ABCB1 signal intensity ABCB1 signal intensity CEBPG 0.81 4.84 x 10-3 CSRNP1 NOT AVAILABLE E 10 kb Hg38 ATF3 0.91 2.64 x 10-4 A E1 E2 B

D K562_R3 H3K27ac

H3K4me1 CD34+ HSPCs DHS

10 kb Hg38

E3

K562_R3 H3K27ac NES 1.7 FDR 0.2% H3K4me1 CD34+ HSPCs Higher expression in HSC/MPP DHS

Figure S9. Adaptive stress signalling and ABCB1 expression across normal hematopoiesis. Related to Figure 6.

(A) Expression of ABCB1, ATF4 and DDIT3 in sorted cord blood populations (26). Data are shown as mean normalised signal intensity ±SEM (n=3-5). (B) Table shows transcription factors upregulated in K562_R1-3 (Figure 2E), their correlation with ABCB1 across normal hematopoiesis and associated p-value. r represents the Pearson product-moment correlation coefficient. (C) Scatter plots show ATF4 or DDIT3 expression versus ABCB1 expression in sorted cord blood populations (26). r represents the Pearson product-moment correlation coefficient. (D) GSEA plot shows the enrichment of genes upregulated in K562_R1-3 (Figure 2C, Table S2) in a list ranked by fold change expression between HSC/MPP and myeloid progenitor cells (CMP, GMP, MEP) (26). (E) ChIPseq tracks for H3K27Ac, H3K4me1 and DNAse-seq (ENCODE) in the region of E1/E2 (top) and E3 (bottom) from normal CD34+ HSPCs. H3K27Ac ChIPseq tracks from K562_R3 are included for reference. K562_R1 K562_R2 K562_R3 Tosedostat Tosedostat 10.4% 7.77% 41.0% 5nMtariquidar Vehicle Vehicle 2.09% 2.99% 16.7% C R3 Vehicle Vehicle Tosedostat Vehicle Tosedostat Vehicle Tosedostat R2 50nMtariquidar R1 Vehicle Vehicle Tosedostat Tosedostat Vehicle Tosedostat Vehicle R3 *** R3 R2 R3 R3 *** R2 5nMtariquidar R1 R3 R3 *** R1 R3 3 2 1 0

R2 Relative ABCB1 MFI MFI ABCB1 Relative Vehicle R2 R2 relative ABCB1 MFI in K562_R1-3 following exposure to 50µM tosedostat or vehicle for 48hrs A R1 Activation of an ISR-like response facilitates escape from ABCB1inhibition. Related Figureto 7. Activationan ISR-likeof response facilitates escape from 0 R2 R2

80 60 40 20

100

% Calcein %

neg

Mean±SEM

B

individualflow histograms eachtheindicatedfor of conditions. (n=3). ***p<0.001 by unpaired t-test. (B) Proportion of cells that are calcein AM negative following K562_R1-3 exposure of to the indicated conditions as determined by flow cytometry (n=3). (C) As for (B), but showing (A) Figure S10. Figure R1

R1

R1 R1 0 1 2 0 1 2 0.5 1.5 2.5 0.5 1.5 2.5 0 1 2 0 1 2 0.5 1.5 2.5 0.5 1.5 2.5 Table S3. ENCODE data used for analyses. Related to Figures 3, 4, 6, S2, S4, S8 & S9.

Upper table shows ChIPseq data from ENCODE used to assess transcription factor (TF)

binding to enhancers in K562 cells (14). The availability of data for all of the TFs upregulated

in resistant K562s (Figure 2E) is shown. *peak not contained within region of enhancer

acetylation but within 1 kb. Lower table shows additional ENCODE data used to define

ABCB1 locus structure and enhancer usage in ABCB1 expressing tissues (14).

Evidence of TF binding TF Cell type Lab Experiment number E1 E2 E3 Promoter E4 ATF4 K562 Michael Snyder, Stanford ENCSR145TSJ Wk YES YES*

JUN K562 Michael Snyder, Stanford ENCSR000EFS YES

JUNB K562 Richard Myers, HAIB ENCSR795IYP YES

XBP1 Unavailable

KLF6 Unavailable

DDIT3 Unavailable

CEBPB K562 Michael Snyder, Stanford ENCSR000EHE YES YES

KLF10 Unavailable

FOSB Unavailable

CEBPG K562 Michael Snyder, Stanford ENCSR490LWA YES

CSRNP1 Unavailable

ATF3 K562 Michael Snyder, Stanford ENCSR028UIU Wk YES YES*

TAL1 K562 Michael Snyder, Stanford ENCSR000EHB YES

GATA2 K562 Kevin White, UChicago ENCSR000DKA YES YES

JUND K562 Michael Snyder, Stanford ENCSR000EGN YES YES

EP300 K562 Michael Snyder, Stanford ENCSR000EGE YES YES

Track Cell type Lab Experiment number

CTCF K562 Bradley Bernstein, Broad ENCSR000AKO CTCF K562 Michael Snyder, Stanford ENCSR000EGM

CTCF K562 Richard Myers, HAIB ENCSR000BPJ

SMC3 K562 Michael Snyder, Stanford ENCSR000EGW

DNase-seq K562 John Stamatoyannopoulos, UW ENCSR000EOY

DNase-seq K562 John Stamatoyannopoulos, UW ENCSR000EOT

H3K27ac Adult liver Bradley Bernstein, Broad ENCSR230IMS

H3K27ac Adult adrenal gland Bradley Bernstein, Broad ENCSR189QAD CD34+ haematopoietic H3K27ac Bradley Bernstein, Broad ENCSR891KSP progenitor

1 CD34+ haematopoietic H3K4me Bradley Bernstein, Broad ENCSR691CSS progenitor

Table S4. Primary AML samples. Related to Figures 6, S6 & S7.

Clinical characteristics of AML patient samples used for assessment of ABCB1 and ATF4

expression (Figure 6A). Samples used for ChIPseq are highlighted in green. Samples used

for daunorubicin exposure experiments are highlighted in red (Figures 6F, S6 & S7). Samples

used for ChIP-PCR are highlighted in blue (Figure 6G). *at diagnosis; PB = peripheral blood;

BM = bone marrow; sl = stemline; sdl = sideline; der = derivative; t = translocation; del =

deletion; add = additional material of unknown origin; idem = denotes the stemline karyotype

in a subclone; inv = inversion; cp = composite karyotype.

Disease ID Sample Gender Age* Blast % Cytogenetics* Status

14 T10 PB Relapse M 52 100 46,XY [20] 53 T1 PB Relapse M 74 97 46,XY [20]

54 T2 PB Refactory F 45 99 47,XY,+11[1]/48,sl,+8[7]/49,sdl,+4[2]

140 T4 PB Relapse M 22 91 46,XY [20] 47,XY,+11[1]/48,sl,+8[7]/49, 148 T7 PB Relapse M 72 80 sdl,+4[2] 160 T9 PB Relapse F 71 97 46,XX [20] 46,XY,t(6;11)(q27;q23)[10]/ 161 T30 PB Relapse M 40 91 48,idem,+der(6)t(6;11),+21[4] 46,XX,t(9;11)(p22;q23),der(21;22)(q10;q10), 171 T3 PB Relapse F 26 89 +der(21;22)[cp10] 225 T15 PB Relapse F 66 96 46,XX [20] 225 T2 PB Relapse F 57 97 46,XX [20]

310 T2 PB Relapse F 73 85 46,XX [20]

338 T2 PB Relapse F 73 89 46,XX [20] 338 T4 PB Refactory F 66 86 46,XX [20]

355 T15 BM Relapse M 73 92 46,XY[20]

355 T22 PB Relapse M 73 95 46,XY[20] 380 T1 PB Relapse M 66 92 46,XY [20]

485 T10 PB Relapse M 66 87 46,XY [20]

497 T2 PB Relapse M 15 97 46,XY,t(8;21)(q22;q22)

546 T6 PB Relapse F 77 82 46,XX [20]

556 T1 BM Relapse M 47 100 46,XX [20]

556 T2 PB Relapse M 47 95 46,XY [20]

572 T11 PB Relapse M 60 95 46,XY [20]

727 T1 PB Relapse M 37 95 Inv(16)

764 T2 BM Presentation F 73 45 Failed

773 T2 PB Presentation M 50 22 46,XY [20]

782 T2 PB Presentation F 34 92 46,XX,t(9;11)(p21.3;q23)[10]

667 T2 BM Relapse F 59 60 del(5q) 47,XY,+13[7]/49- 893 T1 PB Relapse M 72 22 51,idem,+8,+9,+14,+15[cp10]/46,XY[13] 889 T2 BM Relapse M 74 97 46,XY [20]

889 T1 PB Relapse M 74 97 46,XY [20]

901 T1 PB Relapse M 69 100 46,XY [20] 946 T2 PB Presentation M 64 73 46,XY,t(6;9)(p22;q34)

953 T2 PB Presentation M 53 >90 46,XY,inv(16)(p13q22)[15]/46,XY[5]

Table S5. UPL primers and probes used for quantitative PCR.

UPL primers and probes used for quantitative PCR

Gene Species Primer Probe

F: ATTGGCAATGAGCGGTTC ACTB Human 11 R: GGATGCCACAGGACTCCAT

F: GGAAATTTAGAAGATCTGATGTCAAAC ABCB1 Human 65 R: ACTGTAATAATAGGCATACCTGGTCA

F: TGGTCAGTCCCTCCAACAAC ATF4 Human 88 R: CTATACCCAACAGGGCATCC

F: CAGAGCTGGAACCTGAGGAG DDIT3 Human 9 R: CCATCTCTGCAGTTGGATCA

F: CTGGAGAGCTCGGACTGC DDIT4 Human 56 R: TCCAGGTAAGCCGTGTCTTC

F: CGCTTACCTCGGCTACCA CEBPB Human 74 R: ACGAGGAGGACGTGGAGAG

Table S6. UPL primers and probes used for ChIP PCR.

UPL primers and probes used for ChIP PCR

Target Species Primer Probe

F: TGGAGGGTCCAGGTAAAAGA E3_1* Human 12 R: GGTCTTGCTCTAGGGTCTGC

F: GCCTGACCTTCCTGACACAC E3_2* Human 89 R: TGCCAGCACTTGGTCTTTTA

F: GGGCCTATTAGAGGGTGGAG C1 Human 52 R: CCAAGTATTAAGCCTAGTACCCATTAG

F: TGCAATAGAACTCCGTAGTAATTGA E3* Human 69 R: CAGTTTTCTATGACCTCACACCA

*E3_1 and E3_2 were used to assess H3K27 acetylation whilst E3 was used to assess

transcription factor binding.

Table S7. Primer sequences used for CRISPR-dCas9-KRAB enhancer silencing.

Name Primers Strand Name Primers Strand

F: CACCGCCTAATTTGGGTTAGTTCCG F: CACCGCCTACTTATAAGTGAAAACG E1_1 E3_7 R: AAACCGGAACTAACCCAAATTAGGC + R: AAACCGTTTTCACTTATAAGTAGGC - F: CACCGCTGGCCGGCACCTACTGCCG F: CACCGTGTTTCCCAGAACTCTATGC E1_2 E3_8 R: AAACCGGCAGTAGGTGCCGGCCAGC - R: AAACGCATAGAGTTCTGGGAAACAC + F: CACCGAACGCAAACTGATCTAGTGA F: CACCGAGAACTAATATGATGTATCA E1_3 E3_9 R: AAACTCACTAGATCAGTTTGCGTTC + R: AAACTGATACATCATATTAGTTCTC - F: CACCGAGCACTAAAGTAGGAGACAA F: CACCGTGCTCTAGGGTCTGCTTAGG E1_4 E3_10 R: AAACTTGTCTCCTACTTTAGTGCTC - R: AAACCCTAAGCAGACCCTAGAGCAC - F: CACCGTCATCTTACTTGTAACCAGC F: CACCGTCCATTCTGCTTTCTCTAAC E1_5 E3_11 R: AAACGCTGGTTACAAGTAAGATGAC + R: AAACGTTAGAGAAAGCAGAATGGAC + F: CACCGCTTGGAACTAATAAGGTTAT F: CACCGATACATTAGACCTTTCTACC E2_1 E3_12 R: AAACATAACCTTATTAGTTCCAAGC - R: AAACGGTAGAAAGGTCTAATGTATC + F: CACCGTCCAGGTTTATACCCCTGGG F: CACCGCAGTTTGGGCCAATGCCATA E2_2 E3_13 R: AAACCCCAGGGGTATAAACCTGGAC + R: AAACTATGGCATTGGCCCAAACTGC - F: CACCGAACTAGGAAAGTAAACCTAT F: CACCGAATACCTACCCTCCACTCTA E2_3 P_1 R: AAACATAGGTTTACTTTCCTAGTTC + R: AAACTAGAGTGGAGGGTAGGTATTC + F: CACCGGGGGCATTCCCCTTTCAAG F: CACCGTAGTAAATGACTCATCACT E2_4 P_2 R: AAACCTTGAAAGGGGAATGCCCCC - R: AAACAGTGATGAGTCATTTACTAC - F: CACCGAGGGAGGCAAGGCTGTAGTC F: CACCGCTGGCTGAAATTAGCTACTG E2_5 P_3 R: AAACGACTACAGCCTTGCCTCCCTC + R: AAACCAGTAGCTAATTTCAGCCAGC + F: CACCGACACACTTATTTTTTAACAA F: CACCGGTGAATGACTAAGAACGGT E3_1 P_4 R: AAACTTGTTAAAAAATAAGTGTGTC + R: AAACACCGTTCTTAGTCATTCACC + F: CACCGTTCCTACCCCTGTCAAAAAC F: CACCGATCTTGAAGGGGACCGCAA E3_2 P_5 R: AAACGTTTTTGACAGGGGTAGGAAC - R: AAACTTGCGGTCCCCTTCAAGATC - F: CACCGCAGCAATGTATACGATTAAC F: CACCGGTCCAGTGCCACTACGGTT E3_3 P_6 R: AAACGTTAATCGTATACATTGCTGC + R: AAACAACCGTAGTGGCACTGGACC + F: CACCGTTACGGTCTGAATACTTAT F: CACCGACATTTGAAAAATAACGTTA E3_4 E4_1 R: AAACATAAGTATTCAGACCGTAAC - R: AAACTAACGTTATTTTTCAAATGTC + F: CACCGAAACAGGACATTGAATAGTC F: CACCGATTACCTCATCATTTAAGA E3_5 E4_2 R: AAACGACTATTCAATGTCCTGTTTC + R: AAACTCTTAAATGATGAGGTAATC + F: CACCGAAAGTAAAAAATTTTGTTCA E3_6 R: AAACTGAACAAAATTTTTTACTTTC +

Table S8. Reagents used for CRISPR guide RNA cloning.

Reagent Volume (µl) A NEB Buffer 2.1 5 Sense oligo 100uM 5 Antisense oligo 100uM 5 ddH20 35 B RE Buffer (3.1) 2 1µg Vector 0.8 BsmBI 1 ddH20 16.2 C T4 ligase buffer 2.5 Annealed oligo 1 T4 Ligase 1.5