Supplementary Table 1. Oligonucleotide primers for RT-PCR to sequence Exon 34
of the Notch-1 PEST domain of the MT-1 cell line
Notch1 Nucleotide
Primer
Gene
Oligo Sequence
Sequence
Application
(NM_017617.3)
Notch1,
Nucleotides 60615’-GTCAACGCCGTAGATGACCT-3’
N692
Exon34
RT-PCR, Sequencing
6080
Notch1,
Nucleotides 70935’-AGCTCATCATCTGGGACAGG-3’
N703
Exon34
RT-PCR, Sequencing
7075
Notch1,
Nucleotides 75055’-CTTACAGATGCAGCAGCAGAACC-3’
N714
Exon34
RT-PCR, Sequencing
7528
Notch1,
Nucleotides 86665’-TGTGTTGCTGGAGCATCTTC-3’
N704
Exon34
Notch1,
N696
RT-PCR, Sequencing
8647
Nucleotides 6809-
5'-CACCTCGTCTCTCCCACCT-3'
Exon34
Notch1,
N716
RT-PCR, Sequencing
6827
Nucleotides 8088-
5'-CCTGGCATCCACAGAGCGCAC-3'
Exon34
Notch1,
N695
RT-PCR, Sequencing
8068
Nucleotides 6338-
Internal Primer,
6357
Sequencing
Nucleotides 7293-
Internal Primer,
7312
Sequencing
Nucleotides 7745-
Internal Primer,
7764
Sequencing
Nucleotides 8331-
Internal Primer,
8310
Sequencing
5'-TGGACGAGTACAACCTGGTG-3'
Exon34
Notch1,
N697
5'-GAGCTTCCTGAGTGGAGAGC-3'
Exon34
Notch1,
N698
5'-CGACCAGAGGAGCCTTTTTA-3'
Exon34
Notch1,
N715
5'-ACAAGCATGCTTGCAAGAAACC-3'
Exon34
1
Supplementary Figure 1 Ex vivo triple combination treatment did not induce
significant apoptosis of PBMCs from normal donors. (A)PBMCs from normal donors
were incubated with diverse agents (2M Compound E, 2nM Bortezomib and 1nM
Romidepsin) for 48 hours. Apoptosis detection was performed by Annexin V/PI staining
and analyzed by flow cytometry. Annexin V+/PI− (lower right quadrant) areas stand for
early apoptotic cells, and Annexin V+/PI+ (upper right quadrant) areas stand for late
apoptotic or necrotic cells. (B) Bar graphs represent the percentages of early apoptotic
cells from normal donors (Annexin V+/PI−) after treatment with therapeutic agents.
Results are expressed as Mean ± SD. N=6.
Supplementary Figure 2 Ex vivo triple combination treatment did not induce
significant apoptosis of PBMCs from ICN-1low ATL patients. (A) PBMCs from ICN1low ATL patients were incubated with diverse therapeutic agents (2M Compound E,
2nM Bortezomib and 1nM Romidepsin) for 48 hours. Apoptosis detection was performed
by Annexin V/PI staining and analyzed by flow cytometry. Annexin V+/PI− (lower right
quadrant) areas stand for early apoptotic cells, and Annexin V+/PI+ (upper right quadrant)
areas stand for late apoptotic or necrotic cells. (B) Bar graphs represent the percentages
of early apoptotic cells from normal donors (Annexin V+/PI−) after treatment with agents.
Results are expressed as Mean ± SD. N=5.
2
Supplementary Information
Materials and Methods
Proliferation assay
ATL cell lines MT-1, ED40515(+) (abbreviated as ED+) , ED40515(-)(abbreviated as
ED-), 43Tb-, LM-Y1, ED41214-, ST-1 as well as the Hut-102 (HTLV-1 infected cell line
from a patient with ATL), Jurkat and HPB-ALL (T-ALL lines), Kit225 (chronic T-celllymphocytic-leukemia) were maintained in RPMI-1640 containing 10% fetal bovine
serum (FBS), 100U/mL of penicillin, and 100μg/mL of streptomycin in an atmosphere
containing 5% CO2. The BJ fibroblast cell line was maintained in DMEM medium
following the recommendation of the ATCC (American Type Culture Collection,
Manassas, Virginia, USA). ATL cells lines were obtained from Dr. Michiyuki Maeda and
were previously described. The other cell lines were obtained from ATCC. Aliquots of
1×104 cells were seeded in 96-well culture plates and incubated with vehicle or
Compound E, RO4929097, or Bortezomib. For Romidepsin, cells were only cultured
with these agents for 6 hours, then cells were washed and cultures continued with media
alone for 42 hours. The cells were pulsed after 48-hours of culture for 6-hours with 1μCi
of [3H]thymidine (GE Healthcare, UK). Then, the cells were harvested with a 96-well
harvester (Tomtec, Hamden, CT, USA) and counted in a β-counter (PerkinElmer Wallac,
Finland).
Patient materials
3
ATL patient peripheral blood samples were obtained from patients under the care of the
Clinical Trials Team, Lymphoid Malignancies Branch, National Cancer Institute (NCI).
This study protocol was approved by the Institutional Review Board of the NCI.
Informed consent was obtained in accordance with the Declaration of Helsinki. All the
ATL patients had circulating antibodies against HTLV-1, initially when recruited.
Furthermore, pathological examination and FACS analysis of materials from these
patients confirmed the diagnosis of ATL.
RT-PCR, sequencing and tranfection
Total RNA from the MT-1 cell line was reverse transcribed using a SuperScript® III
First-Strand Synthesis System (Invitrogen, NY, USA). Oligonucleotide primers for PCR
amplification of exon 34 of the Notch-1 gene are listed in supplementary Table-1. The
amplified PCR product was purified using a QuickStepTM2 PCR-Purification-Kit
(EdgeBio, Gaithersburg, MD, USA) and was directly sequenced using the BigDye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, NY, USA).
Human Notch-1 intracellular domain (hICN-1) expression construct EF.hICN1.CMV.GFP was obtained from Addgene(Cambridge MA, USA). Oligonucleotide
primers used for PCR amplification of the cleaved portion of the Notch-1 gene are:
hICN-1 Forward: 5’-GCC ACC ATG CGG CGG CAG CAT GGC CAG CTC-3’ and
hICN-1 Reverse: 5’-CTT GAA GGC CTC CGG AT GCG GGC-3’. A point mutation
was introduced at nt 7536 (C->T) of the Notch-1 gene using QuikChange II Site-Directed
Mutagenesis Kit (Aligent Technologies, Santa Clara, CA, USA). Oligonucleotide primers
use for the site directed mutagenesis of Notch-1 are: hICN-1_nt7536 (C->T), Forward: 5'-
4
ACC CCT TCC TCA CCC T GTC CCC TGA GTC CCC-3' and hICN-1_nt7536 (C->T),
Reverse: 5'-GGG GAC TCA GGG GAC A GGG TGA GGA AGG GGT-3'. The mutant
hICN-1 clones were sequenced directly using the BigDye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems, NY,USA). The amplified PCR product was
subcloned into the pEF6/V5-His vector and was sequenced dirtectly using the BigDye
Terminator v3.1 cycle sequencing kit (Applied Biosystems, NY,USA). The pEF6/V5-His
vectors containing either the wild type hICN-1 or the mutant hICN-1 were digested with
EcoRV and BamHI and subcloned into pcDNA3.1Myc (His B) vector, then transfected
into 293T cells using the Lipofectamine 200 reagent (Life Technologies, Carlsbad, CA,
USA) according to the manufacture’s instructions.
Western blot analysis
Samples from whole-cell lysates were prepared and proteins (30 μg) were subjected to
Western blot analysis. The blots were probed with a human monoclonal antibody directed
to cleaved Notch-1(Val 1744), -actin (Cell Signaling, Danvers, MA, USA). The protein
bands recognized by the antibodies were visualized with an enhanced chemiluminescence
Western blotting detection system (GE Healthcare, WI, USA).
Therapeutic study
Therapeutic experiments were performed on selected mice with palpable tumors, which
occur 10 to 12 days after tumor inoculation. For the therapeutic study, Compound E was
dissolved in polyethylene glycol-300 (PEG-300,VWR Scientific Products) at 10mol/Kg,
and continuously administered via a subcutaneous mini-osmotic pump (ALZET, CA,
5
USA). Mini-osmotic pumps were implanted on the day treatment was initiated. The miniosmotic pumps delivered vehicle or therapeutic levels of drug at a flow rate of 0.11μL/h
for 28 days. Bortezomib was provided at 0.5mg/kg/injection by intraperitoneal injection
twice per week for 4 weeks. Romidepsin was administrated at 0.5mg/kg by i.p injection
every other day for 4 weeks. The therapeutic groups were set up with one agent, or two or
three agent combinations. One group of mice that received 200 μL PBS weekly for 4
weeks served as a control. There were 10 mice per group. The groups were randomly
assigned and had comparable average levels of tumor size at the beginning of the
therapeutic trials.
Ex vivo cultures of PBMCs from ATL patients
PBMCs from ATL patients were cultured ex vivo in RPMI-1640 with 10% FBS in the
presence of inhibitors. On day 6 of the culture, 3H-TdR was added for the last 6 hours.
Cell proliferation was measured by thymidine incorporation.
6