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Supplementary Table S1: Ibrutinib sensitivity screening with NCI-60 panel

Cell line Time zero Control −8.0 −7.0 −6.0 −5.0 −4.0 GI50 TGI LC50

Leukemia

CCRF-CEM 0.466 1.488 1.449 1.415 1.394 0.630 0.225 3.51E-06

1.72E-05

9.40E-05

HL-60 (TB) 1.254 2.746 2.681 2.837 3.032 2.495 0.397 1.66E-05

3.54E-05

7.57E-05

K-562 0.565 2.417 2.260 2.341 2.039 0.538 0.293 2.24E-06

8.78E-06

>1.00E-4

MOLT-4 0.950 2.450 1.964 2.019 2.186 0.789 0.365 2.12E-06

6.74E-06

5.49E-05

RPMI-8226 0.744 2.310 2.415 2.423 2.271 1.300 0.337 5.84E-06

2.47E-05

8.85E-05

Non–small cell lung cancer

A549/ATCC 0.538 1.603 1.545 1.402 1.426 1.168 0.075 1.16E-05

2.55E-05

5.64E-05

EKVX 0.615 1.315 1.244 1.093 1.104 0.901 0.272 4.82E-06

2.65E-05

8.71E-05

HOP-62 0.415 1.066 1.040 1.002 0.974 0.776 0.060 1.09E-05

2.47E-05

5.59E-05

HOP-92 0.597 0.989 0.911 0.824 0.777 0.509 0.073 4.57E-07

5.71E-06

3.04E-05

NCI-H226 0.760 1.788 1.686 1.625 1.453 1.002 0.214 2.49E-06

1.76E-05

5.89E-05

NCI-H23 0.324 0.929 0.944 0.925 0.930 0.708 0.046 1.23E-05

2.66E-05

5.75E+05

NCI-H322M 0.513 1.433 1.318 0.834 0.700 0.706 0.027 5.16E-08

1.52E-05

4.10E-05

NCI-H460 0.167 1.340 1.398 1.433 1.350 0.730 0.014 9.14E-06

2.21E-05

5.03E-05

NCI-H522 0.808 1.452 1.326 1.265 1.254 1.080 0.100 5.18E-06

2.12E-05

5.13E-05

Colon cancer

COLO 205 0.272 1.070 1.134 1.144 1.153 0.774 0.034 1.22E-05

2.62E-05

5.63E-05

HCC-2998 0.751 1.196 1.234 1.133 1.227 1.006 0.027 1.11E-05

2.36E-05

4.99E-05

HCT-116 0.274 1.726 1.785 1.808 1.765 0.977 0.019 9.36E-06

2.20E-05

4.95E-05

HCT-15 0.299 1.847 1.744 1.614 1.559 1.074 0.086 1.00E-05

2.59E-05

6.68E-05

HT29 0.154 1.002 0.991 0.976 0.887 0.397 0.024 4.26E-06

1.79E-05

4.96E+05

KM12 0.328 1.316 1.391 1.386 1.351 0.471 -0.002 3.99E-06

1.34E-05

3.66E-05

SW-620 0.171 0.871 0.880 0.884 0.873 0.702 0.058 1.52E-05

3.41E-05

7.67E-05

CNS cancer

SF-268 0.399 1.138 1.158 1.123 1.017 0.617 0.019 4.18E-06

1.72E-05

4.33E-05

SF-295 0.563 1.897 1.926 1.683 1.534 1.036 0.117 4.07E-06

2.04E-05

5.56E-05

SF-539 0.667 1.924 1.938 1.949 1.769 0.903 -0.002 3.52E-06

1.44E-05

3.79E-05

SNB-19 0.490 1.460 1.442 1.406 1.363 1.175 0.003 1.32E-05

2.60E-05

5.12E-05

SNB75 0.848 1.479 1.345 1.358 1.418 0.870 0.010 2.91E-06

1.08E-04

3.33E-05

U251 0.297 1.610 1.583 1.473 1.300 0.774 0.034 4.56E- 1.95E+0 4.90E-

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Melanoma

LOX IMVI 0.298 1.921 1.851 1.681 1.466 0.541 0.047 2.43E-06

1.41E-05

4.52E-05

MALME-3M 0.912 2.062 2.181 2.129 2.047 1.712 0.131 1.34E-05

2.80E-05

5.89E-05

M14 0.305 1.049 1.061 1.059 0.990 0.546 0.055 5.06E-06

1.92E-05

5.25E-05

MDA-MB-435 0.356 1.519 1.561 1.406 1.356 0.700 0.018 4.34E-06

1.73E+05

4.36E-05

SK-MEL-2 0.242 0.423 0.357 0.226 0.200 0.111 0.029 1.56E-08

8.00E-08

7.71E-06

SK-MEL-28 0.348 1.058 1.066 1.105 1.066 0.759 0.064 1.14E-05

2.60E-05

5.93E-05

SK-MEL-5 0.413 2.463 2.436 2.390 2.151 1.131 0.007 4.99E-06

1.83E-05

4.34E-05

UACC-257 0.719 1.027 1.011 0.751 0.675 0.492 0.024 3.39E-08

4.26E-07

1.92E-05

UACC-62 0.577 2.169 2.038 1.812 1.617 0.834 0.078 2.05E-06

1.44E-05

4.14E-05

Ovarian cancer

IGROV1 0.393 0.612 0.418 0.284 0.203 0.106 0.026 <1.00E-8

1.96E-08

1.15E-06

OVCA-3 0.301 0.725 0.727 0.648 0.561 0.383 -0.001 1.85E-06

1.45E-05

3.81E-05

OVCAR-4 0.488 1.428 1.505 1.340 1.292 0.951 0.034 9.50E-06

2.22E-05

4.98E+05

OVCAR-5 0.458 1.293 1.222 1.150 1.098 1.067 0.034 1.37E-05

2.76E-05

5.52E-05

OVCAR-8 0.340 1.025 0.967 0.853 0.771 0.485 0.017 2.04E-06

1.52E-05

4.10E-05

NCI/ADR-RES 0.429 1.235 1.224 1.144 0.981 0.709 0.202 3.52E-06

2.49E-05

9.26E-05

SK-OV-3 0.550 1.224 0.799 0.584 0.562 0.601 -0.007 <1.00E-8

1.17E-05

3.43E-05

Renal cancer

786-0 0.574 2.133 2.087 1.889 1.497 0.857 0.210 1.67E-06

1.67E-05

6.85E-05

A498 0.746 1.415 1.373 1.304 1.252 0.686 0.078 2.02E-06

8.00E-06

3.27E-05

ACHN 0.337 1.569 1.469 1.007 0.814 0.562 0.015 1.90E-07

1.45E-05

3.97E-05

CAKI-1 0.639 1.766 1.467 1.126 0.975 0.711 0.068 5.96E-08

1.17E-05

3.88E-05

RXF 393 0.455 0.720 0.684 0.631 0.594 0.367 0.146 1.08E-06

5.36E-06

4.26E-05

SN 12C 0.776 2.682 2.343 1.993 1.779 1.010 0.183 1.16E-06

1.37E-05

5.03E-05

TK-10 0.588 1.280 1.022 0.755 0.647 0.545 0.059 2.13E-08

3.46E-06

3.28E-05

UO-31 0.455 1.152 1.003 0.837 0.715 0.371 0.016 1.86E-07

4.65E-06

2.53E-05

Prostate cancer

PC-3 0.531 0.988 0.976 0.962 0.976 0.621 0.028 4.08E-06

1.49E-05

4.07E-05

DU-31 0.378 1.313 1.269 1.033 0.959 0.680 -0.003 2.55E-06

1.75E-05

4.19E-05

Breast cancer

MCF7 0.252 1.278 1.255 1.226 1.164 0.590 0.038 4.95E-06

1.90E-05

5.05E-05

MDA-MB-231/ATCC 0.431 1.225 1.208 1.122 1.059 0.750 0.146 5.57E-

062.38E-

057.04E-

05 HS 578T 0.627 1.423 1.348 1.280 1.252 0.911 0.516 4.62E- 4.66E- >1.00E-

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BT-549 1.070 1.959 1.959 1.943 1.891 1.204 0.403 3.53E-06

1.56E-05

6.92E-05

T-47D 0.494 1.005 0.846 0.730 0.735 0.477 0.080 6.80E-08

8.51E-06

3.79E-05

MDA-MB-468 0.591 1.373 1.140 0.642 0.436 0.305 0.095 2.08E-08 1.58E-

07 1.10E-05

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Supplementary MaterialsSupplementary Table S2. The sensitivity of cell lines to ibrutinib is related to K-ras status

Cell line Screen GI50 K-ras PTEN loss EGFR p53 Others

NCI-H1975 NCI300 89 nM WT WT T790M/L858R R273H APC: CDKN2A: PIK3CA: myc ampEKVX NCI60-1 79.9 nM WT COR-L 105 NCI300 67.5 nM WT WT WT H322M NCI60-2 51.6 nM WT WT R248L CDKN2AHOP92 NCI60-2 457 nM WT NCI-H596 NCI300 454 nM WT p53/RB PIK3CA: RB1SK-MES-1 NCI300 44.8 nM WT WT WT p53 CDKN2AH322M NCI60-1 34.6 nM WT WT WT R248L CDKN2ANCI-H1993 NCI300 303 nM WT WT WT C242W STK11/LKB1PC-9 NCI300 18.7 nM WT WT 746-750 p53 NCI-H2170 NCI300 137.6 nM WT WT WT R158G CDKN2AA549 NCI300 >1 M G12S WT WT WT STK11/LKB1: CDKN2ACAL-12T NCI300 >1 M WT p53 BRAF: CDKN2ACalu-1 NCI300 >1 M G12C WT WT WT COR-L23 NCI300 >1 M G12V CDKN2ADV-90 NCI300 >1 M Mutated p53 APC: CDKN2AH226 NCI60-2 >1 M WT WT WT WT H23 NCI60-2 >1 M G12C R233* WT M246I STK11/LKB1H460 NCI60-1 >1 M Q61H WT WT WT STK11/LKB1: PIK3KA: CDKN2AH522 NCI60-1 >1 M WT WT WT FS*56 HOP62 NCI60-1 >1 M G12C WT LCLC-103H NCI300 >1 M WT WT p53 CDKN2ANCI-H1651 NCI300 >1 M WT WT WT C176Y CDKN2ANCI-H1693 NCI300 >1 M WT p53 NCI-H1792 NCI300 >1 M G12C p53 NCI-H2122 NCI300 >1 M G12C C176F CDKN2A: CDH1: STK11/LKB1NCI-H358 NCI300 >1 M G12V WT WT WT NCI-H441 NCI300 >1 M G12V WT WT R158L NCI-H810 NCI300 >1 M WT WT WT WT NCI-H838 NCI300 >1 M WT WT WT E63* STK11/LKB1: CDKN2ASK-LU-1 NCI300 >1 M G12D WT Unknown p53 CDKN2AVMRC-LCP NCI300 >1 M WT p53 CDKN2A: STK11/LKB1

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Supplementary Materials

Supplementary Figure Legends

Supplementary Figure S1. Response of MDA-MB-361 cells to multiple inhibitors. MD-MB-361 cells were plated in 96-well plates overnight before treatment with inhibitors for 72 hours. CellTiter-Glo® was used for quantifying cell growth. The assay was repeated 3 times, and the error bars represent SD.

Supplementary Figure S2. Response of BT-474 (A) and SK-BR-3 (B) cells to treatment with different ErbB kinase inhibitors. The cells were treated with inhibitors continuously for 72 hours, and alamarBlue® assay was used to quantify cell growth.

Supplementary Figure S3. Response of cell signaling pathways to treatment with ibrutinib in multiple cell lines. Cells were treated for 1 hour with ibrutinib. 50,000 cells were loaded in each lane.

Supplementary Figure S4. Ibrutinib inhibited the growth of BT-474 xenograft tumors and related signaling pathways in the tumor samples. (A) Inhibition of BT-474 xenograft growth in NOD-SCID mice by ibrutinib administered at once -daily doses of 3 mg/kg (▲) or 16 mg/kg (▼) compared to vehicle control (▀). Significance for differences from control is indicated by *(P<0.05) or **(P<0.01). (B) Comparison of pharmacokinetic parameters among different strains of mice dosed at 16 mg/kg and 48 mg/kg, PO. (C) Selected images for pERK staining from the BT-474 tumors dosed at 16 and 48 mg/kg, PO, and the data analysis. *P<0.05 and **P<0.01. Error bars represent standard deviation (SD). (D) Relative phosphorylation of AKT (pAKT/AKT) for harvested BT-474 tumors as for (C) at different timepoint. The density of each band was normalized to the first band of vehicle. The results for 48 mg/kg were graphed. Error bars represent SD.

Supplementary Figure S5. Ibrutinib irreversibly inhibited SK-BR-3 cells, and bound covalently to EGFR and HER4 in rapid dilution assays. (A) SK-BR-3 cells were treated with ibrutinib at 0.1 and 0.5 µM for varied times before washing twice with fresh media or media with drug, as for no-washout group. Cells were cultured for another 2 hours after washout before harvesting. (B) The growth of SK-BR-3 cells was still inhibited 6 days after original treatment with ibrutinib for 1 hour using wash-out step. Cells were plated in 10 cm plate in triplicate, and washout was repeated twice. The cells were counted with Coulter counter. *P<0.05, compared with control. Potency of irreversible binding to EGFR (C) and HER4 (D) by ibrutinib, afatinib, and neratinib with rapid dilution assays. The value of kobs quantifies the potency of covalent bond formation. BTK was assayed as a comparison (C). The labeled time indicates pre-incubation time for test compound and enzyme.

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