Evaluation of the in Vitro and in Vivo Efficacy of the JAK Inhibitor AZD1480 Against

Evaluation of the in vitro and in vivo efficacy of the JAK inhibitor AZD1480 against JAK-mutated acute lymphoblastic leukemia

Santi Suryani1‡, Lauryn S. Bracken1‡, Richard C. Harvey2, Keith C.S. Sia1, Hernan Carol1, I-Ming Chen2, Kathryn Evans1, Philip A. Dietrich1, Kathryn G. Roberts3, Raushan T. Kurmasheva4, Catherine A.Billups5Charles G. Mullighan3, Cheryl L. Willman2, Mignon L. Loh6, Stephen P.Hunger7, Peter Houghton4, Malcolm Smith8and Richard B. Lock1

1Children's Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, UNSW, Sydney, Australia

2Cancer Center, University of New Mexico, Albuquerque, NM

3Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN

4Center for Childhood Cancer, Nationwide Children’s Hospital, Columbus, OH

5Department of Biostatistics, St. Jude Children's Research Hospital, Memphis, TN

6Department of Pediatrics, University of California at San Francisco, CA

7University of Colorado Denver School of Medicine and Children’s Hospital Colorado, Aurora, CO

8CTEP/NCI, Bethesda, MD

‡ These authors contributed equally to this work.

SUPPLEMENTARY MATERIALS

SUPPLEMENTARY METHODS

Pediatric Preclinical Testing Program scoring method

Mice were categorized as Progressive Disease 1 (PD1) if the %huCD45+ never dropped below 1% and mice reached event before the end of the study period (42 days post-treatment initiation) and the EFS was <1.5-fold that of the control group (Supplementary Table S1). Mice were assigned a score of Progressive Disease 2 (PD2) if they met the criteria for PD1 but the EFS was ≥1.5-foldthat of the control group. Mice were classified as exhibiting Stable Disease (SD) if the %huCD45+ never dropped below 1% but they did not reach event before the end of the study. A Partial Response (PR) was assigned if the %huCD45+dropped below 1% at any one time point regardless of whether an event was reached by the end of the study. A mouse received a score of Complete Response (CR) if the %huCD45+ dropped below 1% for two consecutive weeks, and a score of Maintained Complete Response (MCR) was assigned if the %huCD45+ was below 1% for the last three weeks of the 42-day study window from treatment initiation. These median group responses were also represented as a numerical score, whereby PD1=0, PD2=2, SD=4, PR=6, CR=8 and MCR=10, the median value of the treated group for each xenograft determined the overall score. The median group response scores were also represented using a color code. Xenografts against which the drug was highly active and scored PR, CR or MCR were classified as an objective response and were shaded yellow to red. Intermediate activity (SD) was shaded gray and low activity (PD1 and PD2) was shaded green.

Mice were closely monitored for signs of drug-related toxicity (weightloss, lethargy, ruffled appearance) and euthanized at the first indication of morbidity. Mice were excluded from the study if they developed spontaneous murine thymic lymphomas.

SUPPLEMENTARY TABLES
Supplementary Table S1. QC of Affymetrix U133_Plus_2.0 Data
USI / Sample Type / CEL File / Scale Factor / GAPDH 3' signal (M33197) / GAPDH 3':5' ratio (M33197)
PAKHZT / Parent / 9906_P3H04.CEL / 16.3 / 39393 / 1.4
Xenograft 05 / XRL_05_(HG-U133_Plus_2).CEL / 7.2 / 71577 / 1.5
PAKMZM / Parent / 9906_P4E03.CEL / 20.6 / 35206 / 1.5
Xenograft 02 / XRL_02_(HG-U133_Plus_2).CEL / 10.2 / 61337 / 1.9
PAKRSL / Parent / 9906_P2F06.CEL / 8.9 / 49762 / 1.1
Xenograft 12 / XRL_12_(HG-U133_Plus_2).CEL / 4.4 / 62254 / 1.3
Xenograft 16 / XRL_16_(HG-U133_Plus_2).CEL / 3.7 / 56315 / 1.2
Xenograft 26 / XRL_26_(HG-U133_Plus_2).CEL / 8.2 / 74129 / 2.1
Xenograft 28 / XRL_28_(HG-U133_Plus_2).CEL / 5.6 / 64035 / 1.7
Xenograft 34 / XRL_34_(HG-U133_Plus_2).CEL / 3 / 34928 / 1.4
Xenograft 37 / XRL_37_(HG-U133_Plus_2).CEL / 3.4 / 43072 / 1.3
PAKSWW / Parent / 9906_P1E02.CEL / 10.3 / 65973 / 1.1
Xenograft 04 / XRL_04_(HG-U133_Plus_2).CEL / 9.6 / 87977 / 1.5
Xenograft 07 / XRL_07_(HG-U133_Plus_2).CEL / 8.9 / 91359 / 1.3
Xenograft 09 / XRL_09_(HG-U133_Plus_2).CEL / 4.7 / 65370 / 1.1
Xenograft 13 / XRL_13_(HG-U133_Plus_2).CEL / 3.3 / 51725 / 1
Xenograft 32 / XRL_32_(HG-U133_Plus_2).CEL / 4.5 / 60275 / 1.2
Xenograft 35 / XRL_35_(HG-U133_Plus_2).CEL / 2 / 29656 / 1.1
PAKVKK / Parent / 9906_P1A04.CEL / 13.1 / 50444 / 1.2
Xenograft 19 / XRL_19_(HG-U133_Plus_2).CEL / 4.6 / 63272 / 1.2
PALJCF / Parent / 9906_P3B07.CEL / 30.9 / 36336 / 1.7
Xenograft 03 / XRL_03_(HG-U133_Plus_2).CEL / 11.1 / 88275 / 2
PALJDL / Parent / 9906_P1H04.CEL / 14.9 / 43580 / 1.2
Xenograft 10 / XRL_10_(HG-U133_Plus_2).CEL / 5.7 / 61970 / 1.3
Xenograft 14 / XRL_14_(HG-U133_Plus_2).CEL / 7.3 / 78251 / 1.4
Xenograft 23 / XRL_23_(HG-U133_Plus_2).CEL / 6 / 63176 / 1.2
Xenograft 31 / XRL_31_(HG-U133_Plus_2).CEL / 8.4 / 75662 / 1.6
Xenograft 36 / XRL_36_(HG-U133_Plus_2).CEL / 2.7 / 33348 / 1.1
Xenograft 38 / XRL_38_(HG-U133_Plus_2).CEL / 3.2 / 39776 / 1
PALKTY / Parent / 9906_P2F01.CEL / 15.9 / 61179 / 1.2
Xenograft 21 / XRL_21_(HG-U133_Plus_2).CEL / 7.2 / 82198 / 1.2
PALLSD / Parent / 9906_P2F03.CEL / 27 / 18298 / 1.8
Xenograft 11 / XRL_11_(HG-U133_Plus_2).CEL / 6.1 / 72254 / 1.3
PALNTB / Parent / 9906_P1C01.CEL / 17.1 / 30873 / 1.2
Xenograft 06 / XRL_06_(HG-U133_Plus_2).CEL / 9.5 / 57506 / 1.3
Xenograft 17 / XRL_17_(HG-U133_Plus_2).CEL / 5.9 / 58321 / 1.4
Xenograft 18 / XRL_18_(HG-U133_Plus_2).CEL / 31.5 / 64196 / 3.3
Xenograft 20 / XRL_20_(HG-U133_Plus_2).CEL / 6.1 / 60790 / 1.3
Xenograft 24* / XRL_24_(HG-U133_Plus_2).CEL / 56.4 / 22737 / 1.8
Xenograft 33 / XRL_33_(HG-U133_Plus_2).CEL / 2.9 / 30796 / 1.1
PALTWS / Parent / 9906_P3G02.CEL / 10.5 / 50567 / 1
Xenograft 22 / XRL_22_(HG-U133_Plus_2).CEL / 7.7 / 77571 / 1.6
PAMDKS / Parent / 9906_P1C11.CEL / 10 / 51628 / 1.1
Xenograft 30 / XRL_30_(HG-U133_Plus_2).CEL / 17.7 / 83546 / 1.9
PAMDRM / Parent / 9906_P2A01.CEL / 18.9 / 52036 / 1.5
Xenograft 01 / XRL_01_(HG-U133_Plus_2).CEL / 10.3 / 68493 / 1.4
Xenograft 08 / XRL_08_(HG-U133_Plus_2).CEL / 11.8 / 84715 / 1.2
Xenograft 15 / XRL_15_(HG-U133_Plus_2).CEL / 3.4 / 51275 / 1.1
Xenograft 25 / XRL_25_(HG-U133_Plus_2).CEL / 7.7 / 58576 / 1.9
Xenograft 27 / XRL_27_(HG-U133_Plus_2).CEL / 5.6 / 65016 / 2
Xenograft 29 / XRL_29_(HG-U133_Plus_2).CEL / 5.8 / 67272 / 1.3

*Excluded from subsequent analysis

Supplementary Table S2. Summary of the Objective Response Measure (ORM) scoring methodology

ORM / Abbreviation / Description / OR Score / Heat Map
Progressive Disease 1 / PD1 / - HuCD45+ never drops below 1% in PB
- Event before the end of study
- LGD value of <1.5 times EFS of control group / 0
Progressive Disease 2 / PD2 / - HuCD45+ never drops below 1% in PB
- Event before the end of study
- LGD value of ≥1.5 times EFS of control group / 2
Stable Disease / SD / - HuCD45+ never drops below 1%
- No event before the end of study period / 4
Partial Response / PR / - HuCD45+ below 1% for one week only / 6
Complete Response / CR / - HuCD45+ below 1% for two consecutive weeks / 8
Maintained Complete Response / MCR / - HuCD45+ below 1% for the last three weeks of study / 10

Supplementary Table S3.Correlation of xenograft with parent U133_Plus_2.0 expression

USI / Xenograft / Evaluated / Within 3-fold / % Within 3-fold / Slope / RSQ
Probe Sets
PAKHZT / XRL_05 / 5466 / 4792 / 87.70% / 0.936 / 0.819
PAKMZM / XRL_02 / 5221 / 4961 / 95.00% / 0.989 / 0.854
PAKRSL / XRL_12 / 5311 / 4184 / 78.80% / 0.837 / 0.801
XRL_16 / 5311 / 4007 / 75.40% / 0.725 / 0.771
XRL_26 / 5311 / 4034 / 76.00% / 1.015 / 0.767
XRL_28 / 5311 / 3943 / 74.20% / 0.843 / 0.783
XRL_34 / 5311 / 4443 / 83.70% / 0.515 / 0.775
XRL_37 / 5311 / 4272 / 80.40% / 0.585 / 0.757
PAKSWW / XRL_04 / 5650 / 4733 / 83.80% / 1.138 / 0.872
XRL_07 / 5650 / 4915 / 87.00% / 1.249 / 0.895
XRL_09 / 5650 / 4965 / 87.90% / 0.793 / 0.899
XRL_13 / 5650 / 4684 / 82.90% / 0.626 / 0.851
XRL_32 / 5650 / 4942 / 87.50% / 0.756 / 0.895
XRL_35 / 5650 / 4838 / 85.60% / 0.385 / 0.805
PAKVKK / XRL_19 / 5102 / 4361 / 85.50% / 0.705 / 0.859
PALJCF / XRL_03 / 4781 / 4021 / 84.10% / 0.867 / 0.792
PALJDL / XRL_10 / 5272 / 4544 / 86.20% / 0.771 / 0.825
XRL_14 / 5272 / 4438 / 84.20% / 0.878 / 0.843
XRL_23 / 5272 / 4339 / 82.30% / 0.729 / 0.809
XRL_31 / 5272 / 4323 / 82.00% / 0.905 / 0.838
XRL_36 / 5272 / 4635 / 87.90% / 0.424 / 0.739
XRL_38 / 5272 / 4341 / 82.30% / 0.492 / 0.758
PALKTY / XRL_21 / 5133 / 4587 / 89.40% / 0.926 / 0.884
PALLSD / XRL_11 / 3911 / 3270 / 83.60% / 0.945 / 0.644
PALNTB / XRL_06 / 4800 / 4160 / 86.70% / 1.079 / 0.845
XRL_17 / 4800 / 4237 / 88.30% / 0.794 / 0.831
XRL_18 / 4800 / 4099 / 85.40% / 1.306 / 0.843
XRL_20 / 4800 / 4230 / 88.10% / 0.835 / 0.848
XRL_33 / 4800 / 4303 / 89.60% / 0.436 / 0.761
PALTWS / XRL_22 / 5671 / 5041 / 88.90% / 1.02 / 0.901
PAMDKS / XRL_30 / 5507 / 4844 / 88.00% / 1.18 / 0.842
PAMDRM / XRL_01 / 4824 / 3981 / 82.50% / 0.925 / 0.859
XRL_08 / 4824 / 3934 / 81.60% / 1.052 / 0.843
XRL_15 / 4824 / 3821 / 79.20% / 0.48 / 0.751
XRL_25 / 4824 / 3961 / 82.10% / 0.718 / 0.844
XRL_27 / 4824 / 3623 / 75.10% / 0.682 / 0.79
XRL_29 / 4824 / 3897 / 80.80% / 0.677 / 0.831

Supplementary Table S4.Probe sets where parent expression is always greater than xenograft

Probe Set ID / Symbol / P (T-test) / Xeno_av / Parent_av
202157_s_at / CELF2 / 3.09E-11 / 10915 / 19233
243489_at / --- / 1.94E-06 / 648 / 15561
231597_x_at / --- / 4.63E-08 / 539 / 9710
1555779_a_at / CD79A / 2.09E-15 / 3226 / 7830
210110_x_at / HNRNPH3 / 3.78E-12 / 2072 / 5137
212152_x_at / ARID1A / 1.41E-16 / 2274 / 4523
213757_at / --- / 3.13E-16 / 693 / 3737
214352_s_at / KRAS / 1.57E-14 / 864 / 3680
239296_at / --- / 1.60E-14 / 558 / 3667
244220_at / --- / 2.09E-10 / 564 / 3328
221718_s_at / AKAP13 / 4.41E-14 / 818 / 3286
1569320_at / GPBP1L1 / 3.20E-14 / 581 / 2657
237062_at / --- / 1.87E-14 / 504 / 2522
237330_at / --- / 9.02E-20 / 505 / 2463
229483_at / --- / 3.19E-14 / 596 / 2369
235595_at / ARHGEF2 / 8.43E-12 / 773 / 2308
229115_at / DYNC1H1 / 9.44E-17 / 520 / 2151
236254_at / VPS13B / 6.40E-13 / 571 / 1960
1556682_s_at / --- / 1.05E-16 / 586 / 1918
236931_at / --- / 1.08E-15 / 761 / 1689

Supplementary Table S5.Probe sets where xenograft expression is always greater than parent

Probe Set ID / Symbol / P (T-test) / Xeno_av / Parent_av
217572_at / --- / 8.22E-03 / 75903 / 500
231628_s_at / SERPINB6 / 4.01E-04 / 23880 / 500
202397_at / LOC128322 /// NUTF2 / 7.85E-18 / 8870 / 1165
207436_x_at / --- / 7.99E-11 / 8393 / 1762
202954_at / UBE2C / 7.65E-12 / 5437 / 657
222388_s_at / VPS35 / 5.42E-19 / 3077 / 862
201643_x_at / KDM3B / 3.06E-16 / 2966 / 1254
222424_s_at / NUCKS1 / 3.37E-10 / 2742 / 657
212547_at / BRD3 / 1.49E-18 / 2556 / 1016
222039_at / KIF18B / 4.46E-10 / 2337 / 691
201526_at / ARF5 / 8.78E-16 / 2312 / 913
223060_at / C14orf119 / 4.18E-19 / 2296 / 669
209449_at / LSM2 / 4.02E-18 / 2286 / 659
211048_s_at / PDIA4 / 5.36E-17 / 2225 / 574
218866_s_at / POLR3K / 1.02E-15 / 2191 / 674
224760_at / SP1 / 6.57E-15 / 2188 / 569
218115_at / ASF1B / 2.56E-14 / 2045 / 579
202261_at / VPS72 / 9.48E-16 / 1989 / 605
215905_s_at / SNRNP40 / 3.28E-20 / 1925 / 500
203252_at / CDK2AP2 / 3.35E-10 / 1877 / 573
204649_at / TROAP / 2.46E-12 / 1830 / 541
226050_at / TMCO3 / 4.87E-12 / 1792 / 729
218355_at / KIF4A / 3.15E-07 / 1748 / 532
203214_x_at / CDK1 / 7.67E-10 / 1605 / 517
225156_at / ELOF1 / 6.66E-12 / 1580 / 802
224959_at / SLC26A2 / 8.34E-13 / 1572 / 578

Supplementary Table S6. Complete summary of in vivo AZD1480 single agent responses for individual mice

Study Information / Animal Counts/Status / EFS Evaluation / Response Evaluation
Line Description / Treatment Group / N11 / Nd2 / Ne3 / Na4 / No.ev5 / KM med.6 / Log-rank P-value / EFS T/C / Median RTV.7 / # PD / # PD1 / # PD2 / # SD / # PR / # CR / # MCR / Median Score / Overall Group Response
PAKHZT / Control / 9 / 0 / 1 / 8 / 8 / 10.9 / >25 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / PD
PAKHZT / AZD1480 / 9 / 0 / 0 / 9 / 9 / 23.6 / <0.001 / 2.2 / >25 / 0 / 0 / 9 / 0 / 0 / 0 / 0 / 2 / PD2
PAKRSL / Control / 9 / 0 / 0 / 9 / 9 / 8.0 / >25 / 9 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / PD
PAKRSL / AZD1480 / 9 / 0 / 0 / 9 / 9 / 8.0 / 0.471 / 1.0 / >25 / 0 / 9 / 0 / 0 / 0 / 0 / 0 / 0 / PD1
PALNTB / Control / 8 / 0 / 0 / 8 / 8 / 2.8 / >25 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / PD
PALNTB / AZD1480 / 7 / 1 / 0 / 6 / 5 / 2.3 / 0.682 / 0.8 / >25 / 0 / 4 / 1 / 1 / 0 / 0 / 0 / 0 / PD1
PAKSWW / Control / 9 / 0 / 1 / 8 / 8 / 19.2 / >25 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / PD
PAKSWW / AZD1480 / 10 / 0 / 1 / 9 / 9 / 7.9 / 0.028 / 0.4 / >25 / 0 / 9 / 0 / 0 / 0 / 0 / 0 / 0 / PD1
PALJCF / Control / 7 / 1 / 1 / 5 / 5 / 18.0 / >25 / 3 / 0 / 0 / 0 / 2 / 0 / 0 / 0 / PD
PALJCF / AZD1480 / 9 / 0 / 1 / 8 / 8 / 14.9 / 0.090 / 0.8 / >25 / 0 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / PD1
PAKYEP / Control / 8 / 0 / 0 / 8 / 8 / 10.6 / >25 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / PD
PAKYEP / AZD1480 / 8 / 0 / 1 / 7 / 7 / 19.9 / 0.515 / 1.9 / >25 / 0 / 2 / 5 / 0 / 0 / 0 / 0 / 2 / PD2
PALJDL / Control / 8 / 0 / 0 / 8 / 8 / 7.2 / >25 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / PD
PALJDL / AZD1480 / 8 / 0 / 0 / 8 / 8 / 18.8 / <0.001 / 2.6 / >25 / 0 / 0 / 8 / 0 / 0 / 0 / 0 / 2 / PD2
ALL-4 / Control / 8 / 0 / 0 / 8 / 8 / 7.1 / >25 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / PD
ALL-4 / AZD1480 / 8 / 0 / 0 / 8 / 8 / 7.7 / 0.951 / 1.1 / >25 / 0 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / PD1
ALL-10 / Control / 8 / 0 / 0 / 8 / 8 / 5.0 / >25 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / PD
ALL-10 / AZD1480 / 8 / 0 / 0 / 8 / 8 / 8.4 / 0.163 / 1.7 / >25 / 0 / 3 / 5 / 0 / 0 / 0 / 0 / 2 / PD2
ALL-31 / Control / 8 / 0 / 0 / 8 / 8 / 8.2 / >25 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / PD
ALL-31 / AZD1480 / 8 / 1 / 0 / 7 / 7 / 7.1 / 0.214 / 0.9 / >25 / 0 / 6 / 1 / 0 / 0 / 0 / 0 / 0 / PD1
N11 - number of mice in group
Nd2 - number of mice that experienced death due to toxicity
Ne3 - number of addidtional mice excluded
Na4 - number of mice analyzed
No.ev5 - number of events; an event is defined as ≥25% hCD45 cells in the peripheral blood
KM med.6 - Kaplan-Meier estimate of median days to event. Time to event was estimated using interpolation
EFS T/C - ratio of event free survival of treated compared to control
Median RTV.7 - Median relative tumor volume. Median % hCD45 at the end of the study

Supplementary Table S7. In vitro Combination Indices (CIs) for AZD1480 and selumetinib
Xenograft ID / ED50 / ED75 / ED90 / Average CI
PAKRSL / 0.16 / 0.06 / 0.03 / 0.08 / VERY STRONG SYNERGISM
PALJCF / 0.07 / 0.02 / 0 / 0.03 / VERY STRONG SYNERGISM
PALLSD / 0.17 / 0.12 / 0.09 / 0.13 / STRONG SYNERGISM
PAMDRM / 0.06 / 0.01 / 0 / 0.02 / VERY STRONG SYNERGISM
ALL-10 JAK1/V658L / 0.27 / 0.32 / 0.43 / 0.34 / SYNERGISM
ALL-4 / 0.54 / 1.31 / 3.25 / 1.7 / NEARLY ADDITIVE
ALL-19 / 0.33 / 0.88 / 2.32 / 1.17 / SLIGHT ANTAGONISM
ALL-26 / 0.61 / 1.55 / 3.98 / 2.04 / ANTAGONISM
PALJDL / 0.41 / 0.63 / 1.15 / 0.73 / MODERATE SYNERGISM

Supplementary Table S8.In vivo responses of JAK-mutated ALL xenografts to AZD1480/selumetinib combination treatment

Xenograft ID / Treatment / Median EFS (days) / LGD (days) / Significance (p-value) / Median ORM
Control / AZD1480 / selumetinib
PALLSD / Control / 7.00
AZD1480 / 7.00 / 0.00 / ns / PD1
selumetinib / 7.00 / 0.00 / ns / ns / PD1
Combination / 7.00 / 0.00 / ns / ns / ns / PD1
PAMDRM / Control / 13.40
AZD1480 / 23.40 / 10.00 / ns / PD1
selumetinib / 14.70 / 1.30 / ns / 0.03 / PD1
Combination / 25.20 / 11.80 / ns / ns / <0.01 / PD1
EFS, event-free survival; LGD, leukemia growth delay; ns, not significant; ORM, objective response measure; PD, progressive disease.

Supplementary Table S9.Complete summary of in vivo AZD1480 single agent and AZD1480/selumetinib combination responses for individual mice

Study Information / Animal Counts/Status / EFS Evaluation / Response Evaluation
USI / Treatment Group / Treatment / N11 / Nd2 / Ne3 / Na4 / No.ev5 / # mice censored / KM med.6 / Comparison / Log-rank P-value / EFS T/C / Median RTV.7 / # PD / # PD1 / # PD2 / # SD / # PR / # CR / # MCR / Median Score / Overall Group Response
PALLSD / A / Control / 8 / 0 / 1 / 7 / 7 / 0 / 7 / . / >25 / 7 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / PD
B / AZD1480 / 8 / 0 / 0 / 8 / 8 / 0 / 7 / AB / 1 / 1 / >25 / 0 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / PD1
C / selumetinib / 8 / 0 / 0 / 8 / 8 / 0 / 7 / AC / 1 / 1 / >25 / 0 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / PD1
D / Combination / 8 / 0 / 0 / 8 / 8 / 0 / 7 / AD / 0.5 / 1 / >25 / 0 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / PD1
BC / 1 / 1
BD / 0.5 / 1
CD / 0.5 / 1
PAMDRM / A / Control / 8 / 0 / 0 / 8 / 7 / 1 / 13.4 / . / >25 / 7 / 0 / 0 / 0 / 0 / 0 / 1 / 0 / PD
B / AZD1480 / 8 / 0 / 0 / 8 / 8 / 0 / 23.4 / AB / 0.3 / 1.7 / >25 / 0 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / PD1
C / selumetinib / 8 / 0 / 0 / 8 / 8 / 0 / 14.7 / AC / 0.8 / 1.1 / >25 / 0 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / PD1
D / Combination / 8 / 0 / 0 / 8 / 8 / 0 / 25.2 / AD / 0.2 / 1.9 / >25 / 0 / 8 / 0 / 0 / 0 / 0 / 0 / 0 / PD1
BC / 0 / 0.6
BD / 0.2 / 1.1
CD / 0 / 1.7
N11 - number of mice in group
Nd2 - number of mice that experienced death due to toxicity
Ne3 - number of additional mice excluded
Na4 - number of mice analyzed
No.ev5 - number of events; an event is defined as ≥25% hCD45 cells in the peripheral blood
KM med.6 - Kaplan-Meier estimate of median days to event. Time to event was estimated using interpolation
EFS T/C - ratio of event free survival of treated compared to control
Median RTV.7 - Median relative tumor volume. Median % hCD45 at the end of the study

SUPPLEMENTARY FIGURES

Supplementary Figure S1.Chemical structures of (A) AZD1480 and (B) selumetinib.

Supplementary Figure S2.Relationship between high CRLF2 expression and engraftment potential of the P9906 cohort.(A) The percentage of engrafted mice was calculated based on the number of mice that showed engraftment in the spleen, peripheral blood and bone marrow. The percentage of organ infiltration was calculated based on the proportion of human CD45+ cells in the spleen (B), peripheral blood (C) and bone marrow (D) at time of harvest.

Supplementary Figure S3. Probe set expression by range.A histogram for the normalized Affymetrix U133_Plus_2.0 MAS5 data of the parents and xenografts is shown. The vertical axis indicates the overall frequency of the range among all 54,504 probe sets evaluated. The horizontal axis shows the dimensions of the expression ranges.

Supplementary Figure S4.Heatmap of microarray gene expression data corresponding to the qRT-PCR data shown in Figure 1.Data were created from the average of 65 Affymetrix probe sets corresponding the 24 genes that were in Figure 1. Twelve probe sets (7 ARHGEF12, 1 KIAA1958, 1 CA6, 1 MDFIC, 1 PON2 and 1 TP53INP1) had not values above background for either the xenografts or parent samples and were excluded. Gene expression intensities for the data were created by setting a minimum background expression of 500 and then expressing the average gene results as doublings lower than the EEF2 control gene expression. The maximum difference for any sample was 7.13 doublings (compared to a minimum of 10 for the qRT-PCR data in Figure 1), with the minimum being only 5.2 doublings (37-fold).

Supplementary Figure S5.In vitro cytotoxicity of AZD1480 against ALL xenograft cells. The panel illustrates the in vitro sensitivity of JAK-mutated ALL xenografts (A-G), one JAK wildtypexenograft with a kinase like signature (H) and one T-ALL (I) to single agent AZD1480. Cells were exposed to various concentrations of AZD1480 for 72 h and cell viability was determined by AlamarBlue assay. Each data point represents the mean + SEM of 3 independent experiments.

Supplementary Figure S6.In vivoefficacy of single agent AZD1480 against ALL xenografts. JAK-mutated (A-F), BCR-JAK2 translocated (G) and a T-ALL (H). Results are presented as the %huCD45+ cells in the PBof individual mice over time (left panels) and the proportion of mice remaining event-free (right panels).Gray shading indicates the treatment period. Black line, vehicle control treated mice; black dotted line, AZD1480 treated mice.

Supplementary Figure S7. Time of drug exposure affects synergistic interactions of AZD1480 and selumetinib against JAK-mutated ALL xenografts. PALLSD (A) and PAMDRM (B) cells were exposed to fixed ratio concentrations of AZD1480, selumetinib and AZD1480/selumetinib for the indicated times. Following drug treatment, cells were washed and incubated in drug-free medium. AlamarBlue was added at 72 h post-initiation of drug treatment, and cell viability estimated. Data are representative of two independent experiments.

Supplementary Figure S8.The extent of inhibition of the JAK/STAT and MAPK signaling pathwaysdepends on the length of exposure to AZD1480/selumetinib. PAMDRM cells were exposed to AZD1480 and/or selumetinib (1 µM) for 12 or 72 h. For the 12 h exposure, cells were washed with warm PBS after 12 h and re-incubated with media. All samples were harvested at 72 h post treatment initiation for total protein extraction and immunoblotting.