Download Supplementary Materials and Methods

Supplementary Information.
1. Supplementary Materials and Methods
Derivation of patient derived xenograft (PDX) model LX-36. We used a SCLC PDX
model PNX1001 (LX-36) established in 2009 NexusPharma at FCCC in collaboration with
Champions Oncology (FCCC approved IRB and IACUC protocol #08-22, PI Vladimir Khazak),
from the primary tumor of a 65 year old female progressing after initial treatment with
docetaxel/cisplatin. For experiments described here, tumor tissues from the F3 generation of the
PNX1001/LX-36 PDX mice were minced on ice to 1-2 mm fragments, mixed 1:1 in
RPMI/Matrigel and implanted subcutaneously in both flanks of 25 C-B17.scid mice, using a 18G
needle, and 200 l volume.
Drug administration. Control animals were treated with vehicle (20% Cremophor RH40
/ 80% D5W). All drugs were purchased from the FCCC pharmacy, except that STA-8666
(alternatively known as STA-12-8666) and ganetespib were provided by Synta Pharmaceuticals.
All injections were delivered intraperitoneally (IP), or intravenously (IV) via a retro-orbital (RO)
route or tail vein injection, using a G27 gauge needle, with drugs diluted to mL/kg body weight.
STA-8666 and ganetespib were formulated using 20% Cremophor RH40 /80% D5W, and dosed
once a week at levels from 50-150 mg/kg as indicated in results, IV/RO (STA-8666) and IV/tail
vein (ganetespib). Irinotecan was given at 60 mg/kg dose weekly (IV/RO). Carboplatin and
etoposide were given at 30 mg/kg dose weekly and 8 mg/kg administered on days 1-3 of a
weekly cycle (24 mg/kg weekly) respectively, and topotecan at 12.5 mg/kg weekly IP. Dosing
concentrations were established based on values reported in the literature (1-3) or by empirical
prior testing to establish maximum tolerated dose (MTD).
Quantitative IHC. For cleaved caspase 3, γH2AX, HSP70 and HSP90, expression was
assessed by the Vectra automated multispectral slide analysis system (Perkin Elmer, Waltham,
MA) using a specific protocol designed for the identification of tumor tissue. Specific algorithms
were also created (nuclear and cytoplasmic respectively) for each marker. H-scores were
subsequently used for results analysis.
Kinome Analysis. All tumors were lysed by mortar and pestle in an ethanol/dry ice bath
using a lysis buffer containing 50 mM HEPES (pH 7.5), 0.5% Triton X-100, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 10 mM sodium fluoride, 2.5 mM sodium orthovanadate, 1X protease
1
inhibitor cocktail (Roche), and 1% each of phosphatase inhibitor cocktails 2 and 3 (Sigma). Cell
lysates were sonicated 3 x 30 s and centrifuged for 15 min (13,000 rpm) at 4oC and the
supernatant was collected and syringe-filtered through a 0.45 m CA membrane.
Protein
concentrations were determined using the Pierce BCA Protein Assay Kit and 5 mg of total
protein were passed over the Multiplexed Inhibitor Bead (MIB) columns for protein kinase
enrichment.
Lysates were brought to 1 M NaCl and passed over a column comprised of the pankinase inhibitor-conjugated beads (Purvalanol B (50 l) (Abcam), VI16832 (50 l), PP58 (50 l)
and CTX-029885 (50 l) (MedKoo Biosciences) to isolate protein kinases from the lysates.
Kinase-bound inhibitor beads were washed with high- and low-salt buffers prior to elution in 100
mM Tris-HCl (pH 6.8), 0.5% SDS and 1% BME at high heat. Proteins were reduced and
alkylated
with
DTT
and
iodoacetamide,
methanol/chloroform extraction.
respectively,
followed
by
purification
by
Purified protein pellets were resuspended in 50 l of 100 mM
triethylammonium bicarbonate and digested overnight at 37o C with sequencing-grade modified
trypsin (Promega). For multiplexing, peptides from each experimental replicate were randomly
labeled with a 6-plex TMT isobaric tag by incubating at room temperature in the dark for 1 h
followed by the addition of 5% hydroxylamine for 15 min at room temperature to quench the
reaction. Labeled peptides were purified using PepClean C18 Spin Columns (Thermo Scientific
Peirce) before LC/MS analysis.
For LC/MS Analysis, a nanoACQUITY UPLC (Waters, Milford, MA) equipped with on-line
two dimensional high pH-low pH fractionation system was used for peptide separation. Buffer
A1 (20 mM ammonium formate pH 10) and buffer B1 (100% acetonitrile) were used as mobile
phases for the first high-pH dimension and buffer A2 (0.1% formic acid in water) and buffer B2
(0.1% formic acid in acetonitrile) were used as mobile phases for the second low-pH dimension.
Peptides were automatically loaded (7 l) onto the first dimension C18 column (nanoEase 5 m
XBridge BEH130 C18 column (300 m x 50 mm) (Waters)) with 3% buffer B1. Peptides were
eluted from this first dimension column in 9 fractions ranging from 11 – 65% buffer B1. Each
fraction of eluted peptides were transferred onto a trap column (10K-2D
Symmetry C18 (180 m x 20 mm) (Waters)) followed by a second dimension PepMap RSLC
C18 Easy-SPRAY column (75m x 500 mm, 2 m particle, column temperature 50o C) (Thermo
Scientific). Peptides were eluted from the analytical column with a 4-step gradient for fractions
1 and 2 consisting of 2 min from 5% to 10%, 65 min from 10% to 20%, 10 min from 20% to
32%, 5 min from 32% to 90%, and maintained at 90% for 10 min prior to returning to 5% for 15
2
min before next fraction elution. Fractions 3 – 9 were eluted with a 4-step gradient consisting of
2 min from 5% to 10%, 60 min from 10% o 25%, 10 min from 25% to 32%, 5 min from 32% to
90%, and maintained at 90% for 10 min before returning to 5% 15 min prior to next fraction
elution.
MS and MS/MS data were acquired with a Q Exactive mass spectrometer (Thermo
Scientific) with a nanoESI source voltage of 2.0 kV, ion transfer tube temperature of 225 o C and
S-lens RF level of 65.0. Data dependent acquistion (DDA) was used with a top 15 method. Full
MS scans were acquired by an Orbitrap mass analyzer over a m/z range of 380 – 1600 with a
resolution of 70,000 (m/z 200) and the number of microscans set to 1. The target value was
3.00 +E06 and a maximum injection time of 120 ms. For MS/MS scans the top 15 most intense
peaks were selected for fragmentation in the higher-energy-collisional-dissociation (HCD) cell
following a MS survey scan.
The normalized collision energy was set to 30% and tandem
mass spectra were acquired in the Orbitrap analyzer with a resolution of 17,500 (m/z 200). The
fixed first mass was set to m/z 120.0. The target value was 1.00+E05 and a maximum injection
time of 150 ms. The number of microscans was set to 1 and the ion selection threshold was
1.0+E05 counts. Peptide match and exclude isotopes were turned on. Dynamic exlusion was
set as 90.0 s. Data obtained from the Q Exactive was processed using Proteome Discoverer
(Thermo Scientific) to identify proteins from database searches and quantify changes in binding
of kinases to MIBs.
Whole Exome Sequencing and Analysis. Whole exome sequencing in LX-36 patient derived
xenograft model was performed by Ambry Genetics (Aliso Viejo, CA). The nsSNPs were
analyzed using the prediction tools SIFT (4), MutationTaster (5), and Polyphen-2 (6).
3
2. Supplementary Figure and Table Legends
Figure S1. In vivo response to STA-8666 treatment of NCI-H69 xenografts A. Average body
weight (BW) in drug treatment cohorts, presented as a ratio to initial BW. B – I. Tumor volume
(TV, left) and body weight (BW, right) normalized to starting tumor volume or body weight in
individual mice from indicated treatment groups.
Figure S2. In vivo response to treatment of PDX LX-36 xenografts. Average tumor volume
(TV) normalized to starting tumor volume in LX-36 xenograft mice from indicated treatment
groups before (A) and after (B) switching to STA-8666 at 150 mg/kg. C, D. Body weight (BW)
dynamics in individual LX-36 xenograft mice from indicated treatment groups, expressed in
relation to starting BW.
Figure S3. Representative images of immunohistochemical (IHC) staining of NCI-H69
tumors following drug treatment. Tumors analyzed were obtained from mice treated with
vehicle, irinotecan, or STA-8666 at the doses indicated at 72 hours after a single dose of drug
(A) and 48 hours after a second weekly dose (B). Scale bar: 40 μm.
Figure S4.
Quantitation of immunohistochemical analysis of cleaved caspase 3, (p
phospho, p)H2AX, HSP70, and HSP90 in recurrent tumors, versus transiently treated
tumors. Vehicle, Irino 60, and STA-150 shows data for individual NCI-H69 xenograft tumors
(analyzed in Figure 3E, top panels). R-50 and R-100 reflect individual tumors initially treated
with STA-8666 at 50 or 100 mg/kg, respectively (from analysis shown in Figures S2B, C). The
graph distinguishes drug resistant tumors (reflected by active growth after substitution of STA8666 at 150 mg/kg; red) from very small residual tumors remaining at the site of xenograft
injection (black), at the end of analysis.
Figure S5. Western blot analysis of NCI-H69 and NCI-H187 cell lines. Representative
images of Western blot analysis of NCI-H69 (A) and NCI-H187 (B) cell lysates harvested at
24hr, 48 hr and 72hr after treatment with vehicle, STA-8666 (100, 60, and 33 nM) and irinotecan
(100 and 60 nM). Quantification of protein expression is presented in Figure 5.
Table S1. Mutational profile of LX-36 xenograft model. Summary of genes mutated in the
LX-36 PDX model based on whole exome sequencing, and analysis of mutations with
4
consequences for protein structure or expression. Second column notes amino acid and
nucleotide changes observed. Genes listed in the table were selected for scrutiny because they
were identified as among those annotated as most frequently mutated in SCLC in multiple
recent studies (7-9) and the COSMIC (Catalogue of Somatic Mutations in Cancer) database
(10). Mutation numbers indicate percentage of samples with a DNA-altering event potentially
affecting protein structure or function out of all samples in the respective study. Mutations refers
to function-damaging missense, frameshift, and splice site mutations, translocations,
homozygous deletions, hemizygous losses, copy-neutral losses of heterozygosity (LOH) and
LOH at higher ploidy.
3. Supplementary References.
1.
Houghton PJ, Cheshire PJ, Myers L, Stewart CF, Synold TW, Houghton JA. Evaluation of
9-dimethylaminomethyl-10-hydroxycamptothecin against xenografts derived from adult and
childhood solid tumors. Cancer chemotherapy and pharmacology. 1992;31:229-39.
2.
Nemati F, Livartowski A, De Cremoux P, Bourgeois Y, Arvelo F, Pouillart P, et al.
Distinctive potentiating effects of cisplatin and/or ifosfamide combined with etoposide in
human small cell lung carcinoma xenografts. Clin Cancer Res. 2000;6:2075-86.
3.
O'Connor R, Liu C, Ferris CA, Guild BC, Teicher BA, Corvi C, et al. Anti-B4-blocked ricin
synergizes with doxorubicin and etoposide on multidrug-resistant and drug-sensitive
tumors. Blood. 1995;86:4286-94.
4.
Ng PC, Henikoff S. SIFT: Predicting amino acid changes that affect protein function.
Nucleic Acids Res. 2003;31:3812-4.
5.
Schwarz JM, Cooper DN, Schuelke M, Seelow D. MutationTaster2: mutation prediction for
the deep-sequencing age. Nature methods. 2014;11:361-2.
6.
Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method
and server for predicting damaging missense mutations. Nature methods. 2010;7:248-9.
7.
Rudin CM, Durinck S, Stawiski EW, Poirier JT, Modrusan Z, Shames DS, et al.
Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in smallcell lung cancer. Nat Genet. 2012;44:1111-6.
8.
Peifer M, Fernandez-Cuesta L, Sos ML, George J, Seidel D, Kasper LH, et al. Integrative
genome analyses identify key somatic driver mutations of small-cell lung cancer. Nat
Genet. 2012;44:1104-10.
5
9.
George J, Lim JS, Jang SJ, Cun Y, Ozretic L, Kong G, et al. Comprehensive genomic
profiles of small cell lung cancer. Nature. 2015;524:47-53.
10. Forbes SA, Bhamra G, Bamford S, Dawson E, Kok C, Clements J, et al. The Catalogue of
Somatic Mutations in Cancer (COSMIC). Curr Protoc Hum Genet. 2008;Chapter 10:Unit 10
1.
6