Supplementary Information
Frequent loss of RAF kinase inhibitor protein expression in acute myeloid leukemia
Armin Zebisch M.D.1-3, Albert Wölfler M.D.1, Isabella Fried M.D.1, Olivia Wolf1, Karin Lind1,
Claudia Bodner1, Martina Haller4, Astrid Drasche4, Daniela Pirkebner4, David Matallanas Ph.D.2,
Oliver Rath Ph.D.3, Karen Blyth Ph.D.3, Ruud Delwel Ph.D.5, Erdogan Taskesen Ph.D.5, Franz
Quehenberger Ph.D.6, Walter Kolch M.D.2,*, Jakob Troppmair Ph.D.4,* and Heinz Sill M.D.1,*
1
Division of Hematology, Medical University of Graz, Graz, Austria; 2Systems Biology Ireland,
University College Dublin, Dublin, Ireland; 3The Beatson Institute for Cancer Research, Glasgow,
United Kingdom; 4Daniel Swarovski Research Laboratory, Innsbruck Medical University, Innsbruck,
Austria;
5
Department of Hematology, Erasmus University Medical Center, Rotterdam, The
Netherlands and 6Institute of Medical Informatics, Statistics and Documentation, Medical University
of Graz, Graz, Austria
*WK, JT and HS contributed equally to this work
Present address corresponding author:
Heinz Sill, M.D., Division of Hematology, Medical University of Graz, Auenbruggerplatz 38, 8036
Graz, Austria. T: +43-316-385-80257, F: +43-316-385-14087, e-mail: heinz.sill@medunigraz.at
Supplementary Table 1
TaqMan® Gene Expression Assays
Gene
Assay number
RKIP
GAPDH
HPRT
Hs00831506_g1 (Applied Biosystems)
Hs03929097_g1 (Applied Biosystems)
Hs01003267_m1 (Applied Biosystems)
RAS sequencing primers
Primer name
Primer sequence 5´- 3´
Annealing
temperature (°C)
NRAS 12/13 fw
NRAS 12/13 rev
NRAS 61 fw
NRAS 61 rev
KRAS 12/13 fw
KRAS 12/13 rev
KRAS 61 fw
KRAS 61 rev
GATGTGGCTCGCCAATTAAC
TCCGACAAGTGAGAGACAGG
TGGGCTTGAATAGTTAGATGC
AGTGTGGTAACCTCATTTCCC
CGATACACGTCTGCAGTCAAC
TATCAAAGAATGGTCCTGCAC
TGCACTGTAATAATCCAGACTGTG
ATGCATGGCATTAGCAAAGACT
60°
60°
62°
62°
62°
62°
62°
62°
Product size
(bp)
221
372
351
300
Supplementary Table 2
Mechanisms of RKIP silencing. The complete coding region of RKIP (exons 1-4) of all
patient samples with RKIP loss (n=19) was sequenced and the DNA copy number assessed.
Neither inactivating mutations nor gene deletions were detected. To screen for methylation of
the RKIP promoter, methylation-specific PCR of a CpG-rich cluster was performed. Ten of
19 samples with RKIP loss demonstrated partial methylation, however, the same pattern was
also detected in AML samples with normal RKIP expression (data not shown). Therefore,
RKIP promoter methylation was excluded as a cause for loss of protein expression. Finally,
we analyzed cDNA expression of Snail – a known transcriptional repressor of RKIP1 – in a
cohort of 285 AML patients,2 but did not detect any correlation to the expression status of
RKIP (data not shown). Wt, Wild-type; NA, not available.
Patient
RKIP sequence
exons 1-4
RKIP DNA copy
number
RKIP promoter
methylation
961
1462
2976
3120
3523
3605
3831
3897
4053
4323
4626
4803
4855
4963
5038
5536
5589
5721
5811
Wt
Wt
Wt
Wt
Wt
Wt
Wt
Wt
Wt
Wt
Wt
Wt
Wt
Wt
Wt
Wt
Wt
Wt
Wt
2
2
2
2
2
2
2
2
2
2
2
2
2
2
NA
2
NA
NA
NA
Absent
Absent
Partial
Partial
Partial
Absent
Absent
Partial
Partial
Partial
Partial
Partial
Partial
Partial
Absent
Absent
Absent
Absent
Absent
Supplementary Figure 1
Densitometry of RKIP protein expression. (a) The graph demonstrates loss of RKIP in four
of seventeen (24%) of AML cell lines. The red line denotes the cut-off for RKIP loss, which
has been set at 25% as outlined in the main manuscript. (b) Density distribution of relative
RKIP expression levels of AML patient samples on a logarithmic scale. Individual
observations are displayed as tick marks on the abscissa. Relative expression values below
25% can be clearly discriminated and were declared to be losses.
Supplementary Figure 2
Basal RKIP expression is not influenced by cytokine levels. (a) 32D cells were starved for 16
hours in RPMI-1640 supplemented with 1% WEHI-conditioned medium as a source of IL-3.
Thereafter, cells were restimulated with up to 5% WEHI-conditioned medium for 5 minutes.
(b) U937 and THP-1 cells were kept in RPMI-1640 containing 10% and 0.05% FCS,
respectively, for 24 hours.
Supplementary Figure 3
RKIP mRNA expression. To address whether loss of the RKIP protein correlates with
reduced mRNA expression in AML, we performed TaqMan® quantitative real-time PCR
from selected AML cell lines (a) and patient samples (b), where sufficient material for RNA
extraction was available (cell lines: RKIP normal, n=5; RKIP loss, n=4; patient samples:
RKIP normal, n=9; RKIP loss, n=10). We used the comparative ∆∆CT method, where RKIP
expression is first normalized to a reference gene (GAPDH) and then compared to a
calibrator. GAPDH was chosen as a reference gene, due to its stable expression in AML cell
lines as proven in quantitative real-time PCR and Western blot analysis. Expression results
could be corroborated by the use of a second reference gene (HPRT; data not shown). Due to
its prominent RKIP expression in Western blot assays, NB-4 was chosen as calibrator with a
value of 100%. Expression of cell lines and patient samples is displayed as % of the
calibrator’s expression. P-values have been calculated using the Mann-Whitney-Wilcoxon
test.
a
b
P=0.027
P=0.007
Supplementary Figure 4
RKIP loss and phosphorylation of ERK. (a,b) The activation of the RAS-MAPK/ERK
pathway, as assessed by the phosphorylation status of ERK is inhibited by (re-)expression of
RKIP. 32D cells, transfected with either FLAG-tagged RKIP or empty vector, were starved in
medium supplemented with 1% WEHI-conditioned medium for 16 hours and subsequently
restimulated with 0 to 5% WEHI-conditioned medium for 5 minutes. THP-1 cells, transfected
with the same constructs, were starved in medium containing 0.05% FCS for 24 hours. (c)
Lysates of AML cell lines were blotted for phosphorylated ERK (pERK) and total ERK.
Densitometric quantification was performed and the ratios pERK/ERK were calculated.
Subsequently, values were normalized to the calibrator sample NB-4 and expressed as x-fold
change. Phosphorylation levels between cell lines with and without RKIP loss were
compared using the Mann-Whitney-Wilcoxon test revealing no significant difference
(P=0.77). Fifty out of 103 AML patient samples used in this study have been characterized
with respect to ERK phosphorylation previously.3 The frequency of patients with constitutive
phosphorylation in the “RKIP loss” vs. “RKIP normal” subgroups was compared using
Fisher’s exact test but did not show a significant difference (P=0.33; data not shown).
Supplementary Figure 5
Transfection of 32D, THP-1 and U937 cell lines. (a) Western blot analysis showing the basal
expression level of RKIP in 32D, U937 and THP-1 cells. Note that all pictures have been
taken from the same blot and have been treated uniformly. (b) Western blots showing the
increase in total RKIP following stable transfection. Membranes have been stained with an
antibody recognizing both endogenous (lower band) and the transfected FLAG-tagged RKIP
(upper band). Due to the strong expression of endogenous RKIP in 32D cells, successful
transfection in these cells could only be visualized by staining with anti-FLAG (see main
manuscript). Note that due to the low expression of RKIP in THP-1, higher concentrations of
both protein and antibodies were used for this blot.
Supplementary Figure 6
Influence of serum starvation on the inhibitory effects of RKIP. Stable transfection of FLAGtagged RKIP and empty vector, respectively, was performed in 32D cells. Subsequently, cells
were maintained in medium supplemented with 1 to 10% WEHI-conditioned medium as
source for IL-3. Viability was measured after 48 hours. For comparison of three independent
experiments, the vector transfected controls have been set at a value of 1 and the relative
decrease of viability in the RKIP transfected cells has been calculated using the ratio viability
RKIP/viability vector. The graphs summarize the results of at least three independent
experiments. Data are expressed as means ± SD and P-values have been calculated using
Student’s t-test. *indicates P<0.050 and **indicated P<0.010.
**
*
100
90
80
% Viability
70
60
Vector
RKIP
50
40
30
20
10
0
1%
2,5%
5%
32D
10%
Supplementary Figure 7
RKIP inhibits oncogenic transformation in the THP-1 AML cells. For soft agar colony
formation assays 100.000 THP-1 cells expressing either FLAG-RKIP or empty vector, were
seeded in soft agar and monitored for growth. On day 28, the percentage of cells growing in
colonies was evaluated. The graphs summarize the results of at least three independent
experiments. Data are expressed as means ± SD and the P-value has been calculated using
Student’s t-test.
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