BERNARD et al
SELECT MICROTUBULE INHIBITORS DISRUPT LYSOSOMES
SUPPLEMENTARY INFORMATION
Select microtubule inhibitors increase lysosome acidity and promote
lysosomal disruption in acute myeloid leukemia (AML) cells
Dannie Bernard1, Marinella Gebbia2, Swayam Prabha1, Marcela Gronda1, Neil MacLean1,
Xiaoming Wang1, Rose Hurren1, Mahadeo A. Sukhai1, Eunice E. Cho1, Morris F. Manolson3,
Alessandro Datti4,5, Jeffrey Wrana4, Mark D. Minden1, Rima Al-Awar6, Ahmed Aman6, Corey
Nislow7, Guri Giaever7 and Aaron D. Schimmer1
AFFILIATIONS: 1Princess Margaret Cancer Centre, University Health Network, Toronto, ON,
Canada; 2Department of Molecular Genetics, Donnelly Centre for Cellular and Biomolecular
Research, University of Toronto, Toronto, ON, Canada; 3Faculty of Dentistry, Dental Research
Institute, University of Toronto, Toronto, ON, Canada; 4Samuel Lunenfeld Research Institute,
Mount Sinai Hospital, Toronto, ON, Canada;
5
Department of Agricultural, Food, and
Environmental Sciences, University of Perugia, Perugia, Italy ; 6Drug Discovery Program,
Ontario Institute for Cancer Research, Toronto, ON, Canada and 7Faculty of Pharmaceutical
Sciences, University of British Columbia, Vancouver, BC, Canada
CORRESPONDING AUTHOR: Aaron D. Schimmer, Princess Margaret Hospital, Rm 9-516,
610 University Ave, Toronto, Ontario, Canada M5G 2M9. Phone: 416.946.2838; Fax:
416.946.6546; E-mail: aaron.schimmer@utoronto.ca.
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SELECT MICROTUBULE INHIBITORS DISRUPT LYSOSOMES
SUPPLEMENTARY MATERIALS AND METHODS
Drugs and reagents
The Natural Product chemical library was purchased from MicroSource Discovery Systems
(Gaylordsville, CT). Deoxysappanone B 7,4’ dimethyl ether (Deox B 7,4) and its reduced form
(Red-Deox B 7,4) were synthesized at the Ontario Institute for Cancer Research (Toronto, ON,
Canada; see supplemental information for synthesis method). The enantiomer separation of
Deox B 7,4 and Red-Deox B 7,4 was outsourced (Lotus Separations, Princeton, NJ).
Nocodazole was obtained from Adipogen (San Diego, CA), vincristine was supplied by Abcam
(Cambridge, MA) and other microtubule inhibitors as well as adriamycin were purchased from
Sigma Aldrich (Oakville, ON, Canada). Bafilomycin A1 was acquired from Cayman Chemicals
(Ann Arbor, MI). All drugs were prepared in dimethyl sulfoxide (DMSO). Unless otherwise
stated, all other chemicals were purchased from Sigma Aldrich (Oakville, ON, Canada).
Cell culture
The leukemia cell lines TEX, OCI-AML2, K562 and THP1 were maintained in Iscove’s
modified Dulbecco’s Medium (IMDM).
maintained in RPMI 1640 medium.
The leukemia cell lines HL60 and U937 were
The ovarian adenocarcinoma cell lines A2780 and
A2780ADR[1], were provided by Dr. Jeremy Squire (Kingston General Hospital, Kingston, ON,
Canada) and were also maintained in RPMI 1640 medium. A2780ADR cells were treated
overnight once a week with 0.1μg/ml adriamycin to maintain the drug resistance phenotype. The
epidermoid carcinoma cell lines KB-3-1 and KB-4.0-HTI36 (a gift from Dr. F. Loganzo, Pearl
River, NY)[2] were grown in Dulbecco’s Modified Eagle Medium (DMEM). For all cell lines
with the exception of TEX, medium was supplemented with 10% fetal bovine serum (FBS),
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SELECT MICROTUBULE INHIBITORS DISRUPT LYSOSOMES
100μg/ml penicillin and 100 U/ml of streptomycin (all from Hyclone, Logan, UT). TEX cells
were grown in the presence of 15% FBS, 100μg/ml penicillin, 100 U/ml of streptomycin, 2mM
L-glutamine, 20ng/ml SCF, 2ng/ml IL-3. All cells were incubated at 37°C in a humidified air
atmosphere supplemented with 5% CO2. For hypoxia experiments, cells were transferred to
hypoxic
culture
chambers
(MACS
VA500
microaerophilic
workstation,
H35
HypoxyWorkStation; Don Whitley Scientific) in which the atmosphere consisted of 5% H2, 5%
CO2 and either 0.2%, 1% or 3% O2 as well as residual N2.
Colony formation assay
The collection and use of human tissue for this study was approved by the local ethics review
board (University Health Network, Toronto, ON, Canada). Peripheral blood samples from
primary AML patients and GM-CSF-mobilized peripheral blood stem cells (PBSCs) from
volunteers donating PBSCs for allotransplant were obtained after informed consent. Blood
samples were subjected to light density isolation with Ficoll-Paque Plus (GE Healthcare,
Uppsala, Sweden) to separate mononuclear cells (MNCs). MNCs (1x105/dish) were plated in
0.1ml IMDM supplemented with 10% FBS and 0.9ml MethoCult GF H4434 medium (StemCell
Technologies, Vancouver, BC, Canada) in duplicate 35mm dishes (Nunclon, Rochester, USA) in
the presence of 100nM Red-Deox B 7,4 or vehicle control. After incubating the dishes for 7
days (AML) or 14 days (normal PBSCs) at 37°C in a humidified air atmosphere supplemented
with 5% CO2, the numbers of AML colonies (CFU-L) containing at least 10 cells and normal
PBSC colonies (BFU-E and CFU-GM) containing more than 100 cells were counted as
previously described[3, 4]. To confirm the type of cells in the colonies, cells were picked when
necessary and stained with May-Grϋnwald-Giemsa as described[3].
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In vivo xenograft studies
Animal studies were carried out according to the regulations of the Canadian Council on Animal
Care and with the approval of the Princess Margaret Cancer Centre ethics review board. Briefly,
SCID mice (Ontario Cancer Institute, Toronto, ON, Canada) were injected subcutaneously with
5x105 OCI-AML-2 cells. Six days post-challenge, when tumors were palpable, mice were
treated 5 days per week with twice-daily intraperitoneal injections of Red-Deox B 7,4 (-)
(75mg/kg) or vehicle control (0.9% NaCl, 10% DMSO and 10% Cremophor EL). Mice were
sacrificed 18 days post-tumor challenge.
Cell cycle analysis
Cells were treated with microtubule inhibitors for the specified times, harvested, washed once in
cold PBS and fixed overnight in cold 70% ethanol at -20°C. Cell were then pelleted, washed
once with PBS and treated with 100ng/ml DNase-free RNase A (Invitrogen, Carlsbad, CA) at
37°C for 30 minutes. A 5μg/ml Propidium Iodine (PI) solution was added prior to measuring
DNA content on a FACScanto II (Becton Dickinson, FL). Results were analyzed with FlowJo
(TreeStar, Ashland, OR).
Mitotic block reversibility assay
Mitotic block reversibility was assessed exactly as previously described[5]. Briefly, U937 cells
were seeded in 75cm2 flasks at 105 cells/ml and allowed to resume log-phase growth.
Concentrations of microtubule inhibitors ranging from 0.25X to 4X their respective IC50 values
for U937 cells were added for 12 hours prior to washing the cells twice with pre-warmed PBS
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SELECT MICROTUBULE INHIBITORS DISRUPT LYSOSOMES
and replenishing with warm, CO2-equilibrated drug-free media. Cells were allowed to recover
for 5 days, with one 80% media replenishment on day 2. Cells were sampled for cell cycle
analysis at the end of the 12 hour drug treatment period as well as 10 hours post-PBS.
Lysosome enrichment
The lysosome enrichment method was adapted from Lardeux et al[6]. Briefly, 108 cells per
condition were harvested, washed once in cold PBS and resuspended in cold 0.25M sucrose
buffer containing 10mM TRIS pH 7.4, 10mM KH2PO4, 0.5mM EDTA and protease inhibitors
(cOmplete Mini tablets; Roche Applied Science, IN). Protease inhibitors were omitted when
isolating lysosomes to assess cathepsin B release. All subsequent steps were carried out at 4°C
and samples kept on ice. Cell homogenates were obtained using a teflon-glass homogenizer
(Glas-Col, Terre Haute, IN) and centrifuged at 3000rpm for 10mins. Supernatants were next
centrifuged at 5000 rpm for an additional 10 minutes. Lysosomes were purified from the
resulting supernatants by centrifugation at 10000rpm for 20 minutes and resuspended in sucrose
buffer supplemented with TRIS. Protein concentrations were determined using the Bradford
reagent (Bio-Rad, Hercules, CA).
V-ATPase gene expression analysis
The expression of V-ATPase genes in the dataset GSE9476 was analyzed. This dataset contains
gene expression data from 26 primary AML samples and 8 normal CD34+ samples[7]. Mean
fold-change for the AML vs. normal CD34+ bone marrow sample comparison was calculated for
all V-ATPase components present on the array (Affymetrix HG U133A GeneChip), and
statistical significance was assessed by ANOVA. Lists of significantly differentially expressed
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genes, including median fold-change and significance values for each gene, were thus derived,
and visualized using a waterfall plot.
REFERENCES
1. Hamilton TC, Young RC, Ozols RF (1984) Experimental model systems of ovarian cancer:
applications to the design and evaluation of new treatment approaches. Semin Oncol
11:285–298.
2. Loganzo F, Hari M, Annable T, et al. (2004) Cells resistant to HTI-286 do not overexpress Pglycoprotein but have reduced drug accumulation and a point mutation in alpha-tubulin. Mol
Cancer Ther 3:1319–1327.
3. Buick RN, Till JE, McCulloch EA (1977) Colony assay for proliferative blast cells circulating
in myeloblastic leukaemia. Lancet 1:862–863.
4. Fauser AA, Messner HA (1979) Identification of megakaryocytes, macrophages, and
eosinophils in colonies of human bone marrow containing neurtophilic granulocytes and
erythroblasts. Blood 53:1023–1027.
5. Towle MJ, Salvato KA, Wels BF, et al. (2011) Eribulin induces irreversible mitotic blockade:
implications of cell-based pharmacodynamics for in vivo efficacy under intermittent dosing
conditions. Cancer Res 71:496–505. doi: 10.1158/0008-5472.CAN-10-1874
6. Lardeux B, Gouhot B, Forestier M (1983) Improved recovery of rat liver fractions enriched in
lysosomes by specific alteration of the sedimentation properties of mitochondria. Anal
Biochem 131:160–165.
7. Stirewalt DL, Meshinchi S, Kopecky KJ, et al. (2008) Identification of genes with abnormal
expression changes in acute myeloid leukemia. Genes Chromosomes Cancer 47:8–20. doi:
10.1002/gcc.20500
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FigS1. Deox B 7,4 promotes apoptosis in various AML cell lines in vitro. Cell death was
determined at 72hrs using AnnexinV and PI staining following treatment of various AML cell
lines with increasing concentrations of Deox B 7,4.
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FigS2. Low oxygen levels increases HIF1 expression in TEX cells
TEX cells were cultured for 24 hours with 21% or 0.2% oxygen.
After incubation, cells were
harvested and HIF1 mRNA expression as measured by Q-RTPCR.
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Supplementary FigS3A. Red-Deox B 7,4 (-) toxicology in vivo (biochemistry). Serum levels
of liver enzymes (AST, ALP), creatine/creatine kinase and bilirubin in SCID mice after 12 days
of Red-Deox B 7,4 (-) administration (75mg/kg intraperitoneally, twice daily, 5 days per week).
Lines represent median.
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Supplementary FigS3B. Red-Deox B 7,4 (-) toxicology in vivo (histology). SCID mice were
treated for 12 days with Red-Deox B 7,4 (-) (75mg/kg intraperitoneally, twice daily, 5 days per
week) or vehicle control. After treatment, mice were sacrificed, organs harvested and stained
with hematoxylin and eosin.
10
V-ATPase activity
(acridine orange fluorescence)
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SELECT MICROTUBULE INHIBITORS DISRUPT LYSOSOMES
Control
4hrs Deox B 7,4
0
8hrs Deox B 7,4
-50
-100
-150
-200
0
60
120
180
Time (secs)
Supplementary FigS4. Deox B 7,4 leads to increased V-ATPase activity. V-ATPase activity
was measured using an acridine orange quenching assay on lysosomes purified from TEX cells
treated for 4hrs or 8hrs with 2.5X the IC50 value for Deox B 7,4 or for 8hrs with DMSO (refer to
Table S2 for IC50 value). Fluorescence was measured every 20secs for 3mins using excitation
and emission wavelengths of 490nm and 540nm, respectively.
Results shown have been
baseline-corrected and are representative of 2 experiments.
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% Lysosomal integrity
Deox B 7,4
R-Deox B 7,4 (-)
Nocodazole
Colchicine
Vinblastine
Vincristine
Paclitaxel
Bafilomycin A1
100
90
80
60
40
20
0
2
4
8
16
24
Time (hrs)
Supplementary FigS5. R-Deox B 7,4 (-) promotes lysosomal disruption in OCI-AML-2 cells
OCI-AML-2 cells were treated with various microtubule inhibitors for the specified times using
concentrations equivalent to 2.5X their respective IC50 values for this cell line (refer to Table S2
for IC50 values). Lysosome integrity was measured using acridine orange staining and flow
cytometry. Bafilomycin A1 was used as a positive control. Only viable cells, based on forward
and side scatter properties, were included in the analysis.
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SELECT MICROTUBULE INHIBITORS DISRUPT LYSOSOMES
10000
***
7500
5000
2500
0
R
-D
eo
C
on
tr
x
ol
B
7,
N
oc 4 ( )
od
az
ol
C
ol
e
ch
ic
Vi
nb ine
B
af las
tin
ilo
e
m
yc
in
A
Tr 1
ito
nX
Cathepsin B release
(relative fluorescence units)
BERNARD et al
Supplementary FigS6. Deox B 7,4 and Red-Deox B 7,4 (-) do not affect lysosome integrity
directly.
Lysosomes were isolated from TEX cells and treated with various microtubule
inhibitors as well as bafilomycin A1 (40μg/sample) for 2hrs before assessing cathepsin B release.
Triton-X detergent was used as a positive control. Drug concentrations used were equivalent to
10X their respective IC50 values for this cell line (refer to Table S2 for IC50 values). Results are
shown as mean  95% CI and are representative of 2 experiments (***p<0.0001 compared to
control cells).
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Supplementary Table S1. List of IC50 values for the various drugs used in this study
Drug
TEX [nM]
OCI-AML-2 [nM]
U937 [nM]*
325.67 ± 11.33
208.33 ± 15.86
554.13 ± 48.44
Red-Deox B 7,4 (-)
Nocodazole
Colchicine
Vinblastine
41.21 ± 2.14
45.17 ± 4.53
9.15 ± 0.45
2.02 ± 0.32
26.60 ± 1.4
46.44 ± 3.75
9.72 ± 0.39
1.04 ± 0.11
328.89 ± 20.88
n/a
5.47 ± 0.25
0.68 ± 0.032
Vincristine
Paclitaxel
4.13 ± 0.25
2.64 ± 0.20
3.54 ± 1.39
0.056 ± 0.06
n/a
n/a
Bafilomycin A1
23.05 ± 1.87
3.71 ± 0.39
n/a
Deox B 7,4
IC50 values represent the mean  95% CI obtained from a minimum of 3 independent experiments. *IC50 values for U937 cells
represent the mean  95% CI obtained from triplicate values of one experiment (n/a: not assessed).
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Supplementary Table S2. Patient characteristics of primary AML samples
Patient
Gender
Age
1
2
3
4
5
M
F
F
M
M
72
74
67
80
62
Disease status
Cytogenetics
Molecular
AML with MDS related changes
AML NOS- AML M5
AML NOS- AML M1
AML NOS- AML M5
AML NOS- AML M5
> 4 abnormalities including -5, -7, -17
XX
XX
Trisomy 8, trisomy 15
XY
Not tested
Not tested
FLT3-ve, NPM-ve
Not tested
FLT3-ve, NPM-ve
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Supplementary Table S3. List of S. cerevisiae screen hits obtained with Deox B 7,4 in glycolytic growth conditions (YPD
medium) and respiratory growth conditions (YPGE medium)
Gene
3.353
Systematic
name
YPL254W
Glycolytic growth conditions (YPD medium)
Standard name
Brief description
HFI1
Histone H2A Functional Interactor
3.226
3.012
YPR135W
YOR153W
CTF4
PDR5
Chromosome Transmission Fidelity
Pleiotropic Drug Resistance
2.942
YDL185W
VMA1
Vacuolar Membrane Atpase
2.881
YPR124W
CTR1
Copper TRansport
2.842
2.785
YLR208W
YLR396C
SEC13
VPS33
SECretory
Vacuolar Protein Sorting
2.661
2.609
2.511
2.135
YDL152W
YKL118W
YLR103C
YGR180C
CDC45
RNR4
Cell Division Cycle
RiboNucleotide Reductase
2.046
YGR252W
GCN5
General Control Nonderepressible
2.043
YOR281C
PLP2
Phosducin-Like Protein
2.018
YKL119C
VPH2
Vacuolar pH
1.931
1.922
1.842
YHR062C
YER155C
YBR118W
RPP1
BEM2
TEF2
Ribonuclease P Protein
Bud EMergence
Translation Elongation Factor
Log2
ratio
Adaptor protein required for structural integrity of the
SAGA complex
Chromatin-associated protein
Plasma membrane ATP-binding cassette (ABC)
transporter
Subunit A of the V1 peripheral membrane domain of VATPase
High-affinity copper transporter of the plasma
membrane
Structural component of 3 distinct complexes
ATP-binding protein that is a subunit of the HOPS and
CORVET complexes
Dubious open reading frame
Dubious open reading frame
DNA replication initiation factor
Ribonucleotide-diphosphate reductase (RNR) small
subunit
Catalytic subunit of ADA and SAGA histone
acetyltransferase complexes
Protein that interacts with the CCT complex to stimulate
actin folding
Integral membrane protein required for V-ATPase
function
Subunit of both RNase MRP and nuclear RNase P
Rho GTPase activating protein (RhoGAP)
Translational elongation factor EF-1 alpha
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1.815
YGL005C
COG7
Conserved Oligomeric Golgi
complex
RecQ Mediated genome Instability
1.814
YPL024W
RMI1
1.811
1.786
1.703
1.699
YDR288W
YJL184W
YOR224C
YOR098C
NSE3
GON7
RPB8
NUP1
Non SMC Element
1.674
YHR083W
SAM35
Sorting and Assembly Machinery
1.656
YNL139C
THO2
1.602
1.591
1.573
1.565
YIL021W
YJL009W
YHR101C
YBL042C
RPB3
suppressor of the Transcriptional
defect of Hpr1 by Overexpression
RNA Polymerase B
BIG1
FUI1
Bad In Glucose
5-FlUorourIdine resistance
1.534
YCR084C
TUP1
1.490
YOR035C
SHE4
deoxyThymidine monophosphate
UPtake
Swi5p-dependent HO Expression
1.452
YKL037W
AIM26
1.446
YLR029C
RPL15A
1.439
1.426
1.420
1.395
YOR340C
YML032C
YNL225C
YLR240W
RPA43
RAD52
CNM67
VPS34
RNA Polymerase B
NUclear Pore
Altered Inheritance rate of
Mitochondria
Ribosomal Protein of the Large
subunit
RNA Polymerase A
RADiation sensitive
Chaotic Nuclear Migration
Vacuolar Protein Sorting
Component of the conserved oligomeric Golgi complex
Subunit of the RecQ (Sgs1p) - Topo III (Top3p)
complex
Component of the SMC5-SMC6 complex
Component of the EKC/KEOPS protein complex
RNA polymerase subunit ABC14.5
FG-nucleoporin component of central core of the
nuclear pore complex
Component of the sorting and assembly machinery
(SAM) complex
Subunit of the THO complex
RNA polymerase II third largest subunit B44
Dubious open reading frame
Integral membrane protein of the endoplasmic reticulum
High affinity uridine permease, localizes to the plasma
membrane
General repressor of transcription
Protein containing a UCS (UNC-45/CRO1/SHE4)
domain
Putative protein of unknown function
Ribosomal 60S subunit protein L15A
RNA polymerase I subunit A43
Protein that stimulates strand exchange
Component of the spindle pole body outer plaque
Phosphatidylinositol (PI) 3-kinase that synthesizes PI-3phosphate
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1.384
YHR024C
MAS2
Mitochondrial ASsembly
1.380
YDL139C
SCM3
1.366
1.363
YDL150W
YMR309C
RPC53
NIP1
Suppressor of Chromosome
Missegregation
RNA Polymerase C
Nuclear ImPort
1.350
1.339
1.328
YOL071W
YLR336C
YOR136W
EMI5
SGD1
IDH2
Early Meiotic Induction
Suppressor of Glycerol Defect
Isocitrate DeHydrogenase
1.308
YAL010C
MDM10
1.304
YDR359C
EAF1
Mitochondrial Distribution and
Morphology
Esa1p-Associated Factor
1.295
1.286
1.257
1.255
1.247
1.237
YPL142C
YDR317W
YER058W
YNL199C
YMR168C
YLL037W
HIM1
PET117
GCR2
CEP3
High Induced Mutagenesis
PETite colonies
GlyColysis Regulation
CEntromere Protein
1.225
YJL209W
CBP1
Cytochrome B mRNA Processing
1.205
1.194
1.184
YBL041W
YLR010C
YGR020C
PRE7
TEN1
VMA7
PRoteinase yscE
TElomeric pathways with STn1
Vacuolar Membrane Atpase
1.170
1.168
YDR381W
YDR331W
YRA1
GPI8
Yeast RNA Annealing protein
GlycosylPhosphatidylInositol anchor
biosynthesis
Larger subunit of the mitochondrial processing protease
(MPP)
Nonhistone component of centromeric chromatin
RNA polymerase III subunit C53
eIF3c subunit of the eukaryotic translation initiation
factor 3 (eIF3)
Subunit of succinate dehydrogenase
Essential nuclear protein
Subunit of mitochondrial NAD(+)-dependent isocitrate
dehydrogenase
Subunit of both the ERMES complex and the SAM
complex
Component of the NuA4 histone acetyltransferase
complex
Dubious open reading frame
Protein of unknown function involved in DNA repair
Protein required for assembly of cytochrome c oxidase
Transcriptional activator of genes involved in glycolysis
Essential kinetochore protein
Dubious open reading frame unlikely to encode a
functional protein
Mitochondrial protein, regulator of COB mRNA
stability and translation
Beta 6 subunit of the 20S proteasome
Protein that regulates telomeric length
Subunit F of the V1 peripheral membrane domain of VATPase
Nuclear polyadenylated RNA-binding protein
ER membrane glycoprotein subunit of the GPI
transamidase complex
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1.163
YOR073W
SGO1
1.162
YNL131W
TOM22
1.158
YNL256W
FOL1
ShuGOshin (Japanese for "guardian
spirit")
Translocase of the Outer
Mitochondrial membrane
FOLic acid synthesis
1.151
1.149
YLR293C
YAL016W
GSP1
TPD3
Genetic Suppressor of Prp20-1
tRNA Processing Deficient
1.140
1.137
1.126
1.109
1.097
1.066
1.063
YFR050C
YBR289W
YDR280W
YDL143W
YMR205C
YLR274W
YPR134W
PRE4
SNF5
RRP45
CCT4
PFK2
MCM5
MSS18
PRoteinase yscE
Sucrose NonFermenting
Ribosomal RNA Processing
Chaperonin Containing TCP-1
PhosphoFructoKinase
MiniChromosome Maintenance
Mitochondrial Splicing System
1.058
1.055
YOR302W
YOL133W
HRT1
1.048
1.047
1.047
1.046
1.040
1.029
YFL037W
YCL017C
YDR045C
YJL001W
YDL092W
YPR132W
TUB2
NFS1
RPC11
PRE3
SRP14
RPS23B
1.028
1.024
YDR232W
YOR209C
HEM1
NPT1
High level expression Reduces Ty3
transposition
TUBulin
NiFS-like
RNA Polymerase C
PRoteinase yscE
Signal Recognition Particle
Ribosomal Protein of the Small
subunit
HEMe biosynthesis
Nicotinate
PhosphoribosylTransferase
1.023
YLR076C
Component of the spindle checkpoint
Component of the TOM (Translocase of Outer
Membrane) complex
Multifunctional enzyme of the folic acid biosynthesis
pathway
Ran GTPase
Regulatory subunit A of the heterotrimeric PP2A
complex
Beta 7 subunit of the 20S proteasome
Subunit of the SWI/SNF chromatin remodeling complex
Exosome non-catalytic core component
Subunit of the cytosolic chaperonin Cct ring complex
Beta subunit of heterooctameric phosphofructokinase
Component of the Mcm2-7 hexameric helicase complex
Nuclear encoded protein needed for splicing of
mitochondrial intron
CPA1 uORF
RING-H2 domain core subunit of multiple ubiquitin
ligase complexes
Beta-tubulin
Cysteine desulfurase
RNA polymerase III subunit C11
Beta 1 subunit of the 20S proteasome
Signal recognition particle (SRP) subunit
Ribosomal protein 28 (rp28) of the small (40S)
ribosomal subunit
5-aminolevulinate synthase
Nicotinate phosphoribosyltransferase
Dubious open reading frame
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1.013
YJL026W
RNR2
RiboNucleotide Reductase
Ribonucleotide-diphosphate reductase (RNR), small
subunit
Mitochondrial oxidoreductase
1.013
YKL195W
MIA40
1.006
YKL059C
MPE1
1.005
YOR358W
HAP5
Log2
ratio
4.102
2.998
Systematic
name
YCR009C
YKL119C
Gene
Mitochondrial intermembrane space
Import and Assembly
Mutant PCF11 Extragenic suppressor Essential conserved subunit of CPF cleavage and
polyadenylation factor
Heme Activator Protein
Subunit of the Hap2p/3p/4p/5p CCAAT-binding
complex
Respiratory growth conditions (YPGE medium)
Standard name
Brief description
RVS161
VPH2
Reduced Viability on Starvation
Vacuolar pH
2.688
YNR036C
MRPS12
2.530
2.528
2.404
2.299
2.181
YPL118W
YDR477W
YLR270W
YGL240W
YNL243W
MRP51
SNF1
DCS1
DOC1
SLA2
Mitochondrial Ribosomal Protein,
Small subunit
Mitochondrial Ribosomal Protein
Sucrose NonFermenting
DeCapping Scavenger
Destruction Of Cyclin B
Synthetic Lethal with ABP1
2.106
YGL115W
SNF4
1.957
1.875
1.818
1.729
1.720
1.656
YOR333C
YPL031C
YJL140W
YDL090C
YJR105W
YKL080W
PHO85
RPB4
RAM1
ADO1
VMA5
Amphiphysin-like lipid raft protein
Integral membrane protein required for V-ATPase
function
Mitochondrial ribosomal protein of the small subunit
Mitochondrial ribosomal protein of the small subunit
AMP-activated serine/threonine protein kinase
Non-essential hydrolase involved in mRNA decapping
Processivity factor
Adaptor protein that links actin to clathrin and
endocytosis
Sucrose NonFermenting
Activating gamma subunit of the AMP-activated Snf1p
kinase complex
Dubious open reading frame
PHOsphate metabolism
Cyclin-dependent kinase
RNA Polymerase B
RNA polymerase II subunit B32
RAS protein and A-factor Maturation Beta subunit of the CAAX farnesyltransferase (FTase)
ADenOsine kinase
Adenosine kinase
Vacuolar Membrane Atpase
Subunit C of the V1 peripheral membrane domain of VATPase
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BERNARD et al
SELECT MICROTUBULE INHIBITORS DISRUPT LYSOSOMES
1.654
YIL134W
FLX1
FLavin eXchange
1.612
YMR188C
MRPS17
1.607
1.588
1.551
1.547
YJR104C
YGL148W
YDR008C
YGL244W
SOD1
ARO2
Mitochondrial Ribosomal Protein,
Small subunit
SuperOxide Dismutase
AROmatic amino acid requiring
RTF1
Restores TBP Function
1.546
YER070W
RNR1
RiboNucleotide Reductase
1.520
YBL099W
ATP1
ATP synthase
1.502
1.476
1.398
YNR025C
YMR077C
YGR020C
VPS20
VMA7
Vacuolar Protein Sorting
Vacuolar Membrane Atpase
1.345
1.307
1.304
YBR171W
YDL006W
YOR209C
SEC66
PTC1
NPT1
1.280
YOR332W
VMA4
SECretory
Phosphatase type Two C
Nicotinate
PhosphoribosylTransferase
Vacuolar Membrane Atpase
1.254
YPR173C
VPS4
Vacuolar Protein Sorting
1.240
YDR359C
EAF1
Esa1p-Associated Factor
1.234
1.227
1.213
YLR038C
YBR026C
YEL027W
COX12
ETR1
VMA3
Cytochrome c OXidase
2-Enoyl Thioester Reductase
Vacuolar Membrane Atpase
Protein required for transport of flavin adenine
dinucleotide (FAD)
Mitochondrial ribosomal protein of the small subunit
Cytosolic copper-zinc superoxide dismutase
Bifunctional chorismate synthase and flavin reductase
Dubious open reading frame
Subunit of RNAPII-associated chromatin remodeling
Paf1 complex
Major isoform of large subunit of ribonucleotidediphosphate reductase
Alpha subunit of the F1 sector of mitochondrial F1F0
ATP synthase
Dubious open reading frame
Myristoylated subunit of ESCRTIII
Subunit F of the V1 peripheral membrane domain of VATPase
Non-essential subunit of Sec63 complex
Type 2C protein phosphatase (PP2C)
Nicotinate phosphoribosyltransferase
Subunit E of the V1 domain of the vacuolar H+-ATPase
(V-ATPase)
AAA-ATPase involved in multivesicular body (MVB)
protein sorting
Component of the NuA4 histone acetyltransferase
complex
Subunit VIb of cytochrome c oxidase
2-enoyl thioester reductase
Proteolipid subunit c of the V0 domain of vacuolar
H(+)-ATPase
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SELECT MICROTUBULE INHIBITORS DISRUPT LYSOSOMES
1.200
1.200
1.189
YDR245W
YGL206C
YER122C
MNN10
CHC1
GLO3
MaNNosyltransferase
Clathrin Heavy Chain
GLyOxalase
1.185
YNL112W
DBP2
Dead Box Protein
1.158
1.151
YDR264C
YMR123W
AKR1
PKR1
1.132
YOR196C
LIP5
AnKyrin Repeat-containing protein
Pichia farinosa Killer toxin
Resistance
LIPoic acid
1.128
YPL078C
ATP4
ATP synthase
1.107
1.095
1.053
1.041
1.024
1.024
YLR260W
YDL206W
YHL025W
YIL060W
YGL168W
YGR105W
LCB5
Long-Chain Base
SNF6
Sucrose NonFermenting
HUR1
VMA21
HydroxyUrea Resistance
Vacuolar Membrane Atpase
1.011
YOR014W
RTS1
Rox Three Suppressor
1.007
YMR058W
FET3
FErrous Transport
Subunit of a Golgi mannosyltransferase complex
Clathrin heavy chain
ADP-ribosylation factor GTPase activating protein
(ARF GAP)
ATP-dependent RNA helicase of the DEAD-box protein
family
Palmitoyl transferase involved in protein palmitoylation
V-ATPase assembly factor
Protein involved in biosynthesis of the coenzyme lipoic
acid
Subunit b of the stator stalk of mitochondrial F1F0 ATP
synthase
Minor sphingoid long-chain base kinase
Putative protein of unknown function
Subunit of the SWI/SNF chromatin remodeling complex
Putative protein of unknown function
Protein of unknown function
Integral membrane protein required for V-ATPase
function
B-type regulatory subunit of protein phosphatase 2A
(PP2A)
Ferro-O2-oxidoreductase
Haplo-insufficiency profiling (HIP) of Deox B 7,4 in S. cerevisiae was performed in either glycolytic growth conditions (YPD
medium) or respiratory growth conditions (YPGE medium). The fitness defects were calculated for each deletion strain as log2 ratios
(mean signal intensity of control/mean signal intensity of drug). Deletion strains with log2 ratio values of more than 1 were
considered hits.
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BERNARD et al
SELECT MICROTUBULE INHIBITORS DISRUPT LYSOSOMES
Supplementary Table S4. Microtubule inhibition by Deox B 7,4 and R-Deox B 7,4 (-) is functionally relevant and overcomes
drug efflux pump-related resistance
Drug
KB-3-1 [nM]
Deox B 7,4
R-Deox B 7,4
Nocodazole
Colchicine
344.46 ± 26.34
89.30 ± 23.32
40.35 ± 3.92
6.14 ± 0.12
KB-4.0-HTI
[nM]
655.93 ± 31.46
149.72 ± 29.83
99.79 ± 10.85
17.57 ± 1.06
Fold resistance
(KB4.0/KB3.1)
1.90
1.68
2.47
2.86
Vincristine
n/a
n/a
n/a
Doxorubicin
n/a
n/a
n/a
A2780 [nM]
300.83 ± 70.23
39.50 ± 0.71
n/a
5.47 ± 0.12
A2780ADR
[nM]
576.17 ± 101.87
146.30 ± 42.19
n/a
324.26 ± 39.14
Fold resistance
(A2780ADR/A2780)
1.92
3.70
n/a
59.28
0.77 ± 0.09
15.59 ± 8.83
845.74 ± 205.67
729.57 ± 148.45
1098.36
46.80
Alpha-tubulin mutated KB-4.0-HTI epidermoid carcinoma cells and their KB-3-1 wild-type controls, as well as A2780ADR ovarian
carcinoma cells overexpressing P-glycoprotein and their A2780 wild-type controls, were treated with increasing concentrations of the
specified microtubule inhibitors for 72hrs. Cell viability was assessed using MTS and data represent IC 50 values ± standard deviation
calculated from 3 individual experiments (n/a: not assessed).sss
23