Supplementary Table S1. Treatment schema for mice using radiotherapy and AZD8055
General treatment schema. Mice received daily AZD8055 administration (via gavage) for 3 weeks and/or 1-2 weeks of 2-Gy daily fractions (Monday-Friday). Doses of each modality varied for the dose escalation phase. For the standard treatment arms, AZD8055 10 mg/Kg and 20 Gy in 10 fractions of radiotherapy were given
.
Experimental Treatment Groups Week 1 Week 2 Week 3 Treatment totals
Untreated Control AZD8055 n/a n/a n/a n/a 10-20 mice XRT n/a n/a n/a AZD8055 Alone AZD8055 ||||||| ||||||| ||||||| 5-20 mg/Kg x 21 days 10-20 mice XRT n/a n/a n/a XRT Alone AZD8055 n/a n/a n/a n/a 10-20 mice XRT ||||| ||||| 20 Gy/ 10 fractions Combined AZD8055 ||||||| ||||||| ||||||| 5-20 mg/Kg x 21 days 10-20 mice XRT ||||| |||||12 Week Obeservation Period 20 Gy / 10 fractions Supplementary Table S2.
Treatment Mean XRT Enhancement Cell line Group Dose Densityb Failuresc/Total Failure Rate Ratiod a XRT Alone 60 Gy/cm³ 14/18 78% Rh30 XRT + 7.33 AZD8055 27 Gy/cm³ 4/15 27% XRT Alone 59 Gy/cm³ 3/12 25% Rh18 XRT + 0.83 AZD8055 44 Gy/cm³ 8/15 53%
a. Radiotherapy b. Given dose, Gy / volume of tumor at initiation of treatment, cc c. Incomplete/no response or recurrence of xenograft after complete response XRT A Mean Dose / XRT A Fraction Failure Free . XRT AZD Mean Dose / XRT AZD Fraction Failure Free where Fraction Failure Free = 1 – (Failures/Total) / / For Rh30: / 7.33 /
/ / For Rh18: / 0.83 /
Supplementary Table S3. Demographics and univariate survival analysis of patients with complete survival data.
Gene Expression Set, n=101 TMA Set, n=94 Median age: 5.6 years Median age: 6.0 years Factor N(%) Mean OS P-value* N (%) Mean OS P-value* female 47 (47) 5.5 38 (40) 5.8 0.167 0.425 male 54 (53) 8.2 56 (60) 6.6
ERMS 36 (36) 8.4 41 (44) 7.3 0.005 0.030 ARMS 65 (64) 6.1 53 (56) 5.5
Non-translocated 56 (55) 8.4 58 (65) 6.8 PAX3/FOXO1 34 (34) 3.6 22 (25) 3.6 PAX7/FOXO1 10 (10) 8.5 <0.001 9 (10) 7.1 0.002 PAX3/NCOA1 1 (1) 2.5 0 (0) - NA - - 5 -
Stage 1 28 (28) 8.9 24 (28) 8.3 Stage 2 10 (10) 4.4 10 (11) 6.9 Stage 3 34 (34) 7.6 <0.001 24 (26) 6.9 <0.001 Stage 4 28 (28) 3.4 29 (31) 3.9 NA - - 7 -
Group I 8 (8) 8.0 1 (1) censored Group II-III 64 (64) 8.5 46 (61) 7.3 <0.001 <0.001 Group IV 29 (28) 3.5 29 (38) 3.8 NA - - 18 -
Abbreviations: TMA (tissue micro-array from Children’s Oncology Group D9902 study); Affy (Affymetrix .CEL files from (Williamson et al., 2010)); Mean OS, mean overall survival in years; NA, not available; ERMS, embryonal rhabdomyosarcoma; ARMS, alveolar rhabdomyosarcoma;
*log-rank
Supplementary Table S4. m-TOR-associated genes identified and used within the gene set enrichment analysis
Pax3 Negative Pax3 Positive RANK RANK GENE RANK IN METRIC RUNNING CORE RANK IN METRIC RUNNING CORE SYMBOL GENE_TITLE GENE LIST SCORE ES* ENRICHMENT GENE LIST SCORE ES* ENRICHMENT restin (Reed-Steinberg cell-expressed RSN intermediate filament-associated 20,268 -0.48 0.02 Yes 20,492 -0.82 0.01 Yes protein) DEPDC6 DEP domain containing 6 15,392 -0.09 -0.20 No 19,807 -0.45 -0.05 Yes v-kit Hardy-Zuckerman 4 feline sarcoma KIT 18,372 -0.21 -0.16 Yes 19,744 -0.44 -0.10 Yes viral oncogene homolog SGK serum/glucocorticoid regulated kinase 18,140 -0.20 -0.19 Yes 19,694 -0.43 -0.14 Yes
RRAGB Ras-related GTP binding B 16,968 -0.14 -0.22 Yes 19,638 -0.42 -0.19 Yes
IRS2 insulin receptor substrate 2 7,811 0.04 0.07 No 19,426 -0.38 -0.22 Yes
TSC22D3 TSC22 domain family, member 3 11,810 -0.02 -0.10 No 19,330 -0.36 -0.26 Yes phosphoinositide-3-kinase, regulatory PIK3R1 17,305 -0.15 -0.22 Yes 18,867 -0.30 -0.28 Yes subunit 1 (p85 alpha) KPNA1 karyopherin alpha 1 (importin alpha 5) 8,705 0.03 0.04 No 18,727 -0.28 -0.30 Yes
AKT1S1 AKT1 substrate 1 (proline-rich) 10,433 0.00 -0.03 No 18,419 -0.25 -0.32 Yes
PRKCA protein kinase C, alpha 19,414 -0.31 -0.06 Yes 17,752 -0.21 -0.31 Yes signal transducer and activator of STAT3 transcription 3 (acute-phase response 18,601 -0.23 -0.08 Yes 17,622 -0.20 -0.33 Yes factor) SMAD, mothers against DPP homolog 3 SMAD3 12,400 -0.03 -0.12 No 17,378 -0.19 -0.34 Yes (Drosophila) GSK3B glycogen synthase kinase 3 beta 7,077 0.06 0.09 No 17,225 -0.18 -0.35 Yes mitogen-activated protein kinase MAPKAP1 10,078 0.01 -0.02 No 17,146 -0.17 -0.37 Yes associated protein 1 MAF1 MAF1 homolog (S. cerevisiae) 5,717 0.08 0.12 No 16,898 -0.16 -0.38 Yes
IRS1 insulin receptor substrate 1 17,373 -0.16 -0.20 Yes 16,558 -0.15 -0.38 Yes
RICTOR - 18,599 -0.23 -0.11 Yes 15,645 -0.12 -0.35 No
TSC2 tuberous sclerosis 2 5,143 0.10 0.13 No 14,200 -0.08 -0.29 No
ULK1 unc-51-like kinase 1 (C. elegans) 13,245 -0.05 -0.15 No 13,853 -0.07 -0.28 No
PRKCH protein kinase C, eta 20,136 -0.44 -0.04 Yes 13,671 -0.07 -0.28 No
GBL - 4,175 0.12 0.13 No 13,108 -0.06 -0.26 No
FKBP8 FK506 binding protein 8, 38kDa 13,641 -0.05 -0.15 No 12,952 -0.05 -0.26 No
UBQLN1 ubiquilin 1 9,391 0.02 0.01 No 12,911 -0.05 -0.27 No phosphatase and tensin homolog PTEN (mutated in multiple advanced cancers 16,856 -0.14 -0.23 Yes 12,893 -0.05 -0.27 No 1) FK506 binding protein 12-rapamycin FRAP1 16,651 -0.13 -0.24 Yes 12,624 -0.05 -0.26 No associated protein 1 eukaryotic translation initiation factor 4E EIF4EBP2 3,313 0.15 0.12 No 12,566 -0.04 -0.27 No binding protein 2 RHEB Ras homolog enriched in brain 8,162 0.04 0.06 No 12,378 -0.04 -0.26 No
SKIP - 17,455 -0.16 -0.19 Yes 12,249 -0.04 -0.26 No v-abl Abelson murine leukemia viral ABL1 13,509 -0.05 -0.16 No 11,722 -0.03 -0.24 No oncogene homolog 1 KIAA0652 KIAA0652 14,824 -0.08 -0.19 No 11,282 -0.02 -0.22 No
PRR5 proline rich 5 (renal) 6,704 0.06 0.10 No 10,751 -0.01 -0.20 No signal transducer and activator of STAT1 18,829 -0.25 -0.06 Yes 9,969 0.00 -0.16 No transcription 1, 91kDa PREX1 - 7,162 0.06 0.09 No 9,747 0.01 -0.15 No
RRAGC Ras-related GTP binding C 13,511 -0.05 -0.15 No 9,477 0.01 -0.14 No ribosomal protein S6 kinase, 70kDa, RPS6KB1 15,393 -0.09 -0.19 No 9,281 0.02 -0.13 No polypeptide 1 guanine nucleotide binding protein (G GNB1L 5,036 0.10 0.12 No 9,238 0.02 -0.13 No protein), beta polypeptide 1-like v-akt murine thymoma viral oncogene AKT1 8,200 0.04 0.06 No 8,958 0.03 -0.12 No homolog 1 FKBP1A FK506 binding protein 1A, 12kDa 13,838 -0.06 -0.16 No 8,794 0.03 -0.11 No tumor protein p53 (Li-Fraumeni TP53 507 0.41 0.03 No 8,583 0.03 -0.11 No syndrome) protein phosphatase 2 (formerly 2A), PPP2CA 10,599 0.00 -0.04 No 8,535 0.03 -0.11 No catalytic subunit, alpha isoform RPS6 ribosomal protein S6 9,529 0.02 0.01 No 8,402 0.04 -0.10 No protein disulfide isomerase family A, PDIA3 14,561 -0.07 -0.18 No 8,328 0.04 -0.10 No member 3 ribosomal protein S6 kinase, 90kDa, RPS6KA1 18,455 -0.22 -0.13 Yes 7,761 0.05 -0.08 No polypeptide 1 TEL2, telomere maintenance 2, homolog TELO2 2,233 0.19 0.12 No 7,328 0.06 -0.07 No (S. cerevisiae) GPHN gephyrin 3,138 0.15 0.11 No 6,814 0.07 -0.05 No
HD huntingtin (Huntington disease) 12,239 -0.03 -0.11 No 6,692 0.07 -0.05 No conserved helix-loop-helix ubiquitous CHUK 2,104 0.20 0.10 No 6,563 0.08 -0.05 No kinase eukaryotic translation initiation factor 4E EIF4EBP1 2,286 0.19 0.14 No 6,397 0.08 -0.05 No binding protein 1 SMAD, mothers against DPP homolog 4 SMAD4 11,212 -0.01 -0.07 No 6,260 0.08 -0.05 No (Drosophila) tyrosine 3-monooxygenase/tryptophan YWHAZ 5-monooxygenase activation protein, 8,922 0.02 0.03 No 6,181 0.08 -0.06 No zeta polypeptide general transcription factor IIIC, GTF3C5 7,600 0.05 0.08 No 6,179 0.08 -0.07 No polypeptide 5, 63kDa EIF4E eukaryotic translation initiation factor 4E 5,692 0.08 0.11 No 5,650 0.10 -0.05 No general transcription factor IIIC, GTF3C2 4,705 0.11 0.13 No 5,357 0.11 -0.05 No polypeptide 2, beta 110kDa general transcription factor IIIC, GTF3C1 5,882 0.08 0.12 No 4,928 0.12 -0.04 No polypeptide 1, alpha 220kDa DEPDC2 DEP domain containing 2 3,707 0.13 0.12 No 4,376 0.14 -0.02 No KH domain containing, RNA binding, KHDRBS1 4,684 0.11 0.12 No 3,524 0.17 0.00 No signal transduction associated 1 PRKCD protein kinase C, delta 18,161 -0.20 -0.17 Yes 3,431 0.17 -0.01 No eukaryotic translation initiation factor 3, EIF3S5 6,363 0.07 0.11 No 3,280 0.18 -0.02 No subunit 5 epsilon, 47kDa BCL2L11 BCL2-like 11 (apoptosis facilitator) 1,167 0.28 0.12 No 2,559 0.22 -0.01 No retinoblastoma 1 (including RB1 4,100 0.12 0.12 No 2,269 0.24 -0.02 No osteosarcoma) RBL1 retinoblastoma-like 1 (p107) 540 0.41 0.07 No 2,173 0.24 -0.04 No
PCNA proliferating cell nuclear antigen 802 0.33 0.10 No 1,176 0.35 -0.02 No
Abbreviations: ES (enrichment score)