Supplemental Information
Table S1. Lists of antibodies used in this study.
For Flow Cytometry
Antibody
CD90.1 (Thy-1.1)-Biotin
Streptavidin-APC
CD13-PE
CD14-PE
CD33-PE
CD34-APC
CD19-APC
CD11b/Mac-1-APC
CD45-PECy7
Company
BD Biosciences
BD Biosciences
BD Biosciences
BD Biosciences
BD Biosciences
BD Biosciences
BD Biosciences
BD Biosciences
BD Biosciences
For Immunoprecipitation and Western Blotting
Antibody
Company
CBL
Cell Signaling Technology
CBL
Cell Signaling Technology
Phospho-CBL (Tyr700)
Cell Signaling Technology
UBASH3B/Sts-1
Abcam
Phospho-Tyrosine
Cell Signaling Technology
RUNX1
Cell Signaling Technology
Phospho-Stat5 (Thy694)
Cell Signaling Technology
Stat5
Cell Signaling Technology
Phospho-Erk1/2 (Thy202/Tyr204)
Cell Signaling Technology
Erk1/2
Cell Signaling Technology
Phospho-Src Family (Tyr416)
Cell Signaling Technology
c-Kit
Cell Signaling Technology
MPL
Amgen
Tubulin
Sigma-Aldrich
Actin
Sigma-Aldrich
Clone#
OX-7
Streptavidin
WM15
M5E2
WM53
581
HIB19
ICRF44
HI30
Product #
554896
554067
555394
555398
555450
555824
555415
550019
557748
Clone#
C49H8
D4E10
D16D7
polyclonal
P-Tyr-100
D33G6
D47E7
polyclonal
197G2
polyclonal
D49G4
D13A2
1.75.6
D-66
AC-74
Product #
2179
8447
8869
ab34781
9411
4336
4322
9363
4377
9102
6943
3074
T0198
A2228
Table S2. Comparison of UBASH3B expression by CBL mutation status in Younger
(<60y) de novo CBF patients
n=131
UBASH3B probe sets
CBL mutated
CBL wild-type
(n=14)
(n=117)
P-value
6.33
(6.02, 6.46)
6.25
(5.86, 6.89)
0.41
5.92
(4.60, 6.71)
5.95
(4.89, 7.88)
0.77
6.42
(5.03, 6.99)
5.94
(4.81, 7.24)
0.002
8.33
(6.98, 9.34)
7.79
(5.76, 9.25)
0.008
8.81
(7.07, 9.24)
8.27
(6.02, 9.63)
0.02
240387_at
Median
Range
244068_at
Median
Range
228353_x_at
Median
Range
228359_at
Median
Range
238587_at
Median
Range
238462_at
0.04
Median
7.58
7.01
Range
(6.07, 9.03)
(4.34, 9.43)
CBF-AML patients were enrolled on one of the following Cancer and Leukemia Group
B (CALGB) frontline treatment protocols: 8525 (n=1)(Ref. 1), 9222 (n=14)(Ref. 2), 9621
(n=61) (Ref. 3), or 19808 (n=55) (Ref. 4). All patients provided written informed
consent. Study protocols were in accordance with the Declaration of Helsinki and
approved by institutional review boards at each center. Microarray data is from
Affymetrix HG-U133 Plus 2.0. P-values for continuous variables are from Wilcoxon
rank sum test. P-values are not adjusted for multiple comparisons.
Supplemental Discussion
UBASH3B/Sts-1 expression in core binding leukemia with/without CBL mutations
We examined the expression of UBASH3B/Sts-1 probe sets (U133 plus2.0; log2 units) in 131 patients
with de novo core-binding factor (CBF) leukemia with/without CBL mutations [41 t(8;21) patients
and 90 inv(16) patients]. UBASH3B/Sts-1 contains an N-terminal Ubiquitin-association (UBA)
domain and a central Src-homology 3 (SH3) domain5. It binds CBL through the SH3 domain and to
ubiquitin via UBA domain. In addition to full-length transcript of UBASH3B/Sts-1, there is a
predicted shorter transcript of UBASH3B/Sts-1 (NCBI Reference Sequence: XM_005271712.2)
lacking exons 1-3. This N-terminal splice variant of UBASH3B/Sts-1 does not have the UBA domain.
Among the six probes for UBASH3B/Sts-1, four probes (228353_x_at, 228359_at, 238587_at,
238462_at) are predicted to detect both full-length and the shorter variant of UBASH3B/Sts-1, while
the other two probes (240387_at, 244068_at) are predicted to detect only full-length UBASH3B/Sts-1.
As shown in Table S2, four probes detecting both full-length and the shorter variant showed
significantly higher levels (around a one log2 difference or ~2-fold difference in expression) of
UBASH3B/Sts-1 in CBL-mutated cases compared to cases without CBL mutations. In contrast, the
two probes, which detect only full-length transcript, showed similar levels of UBASH3B/Sts-1
expression. These data suggest that the N-terminal splice variant of UBASH3B/Sts-1 is specifically
upregulated in CBL-mutated CBF leukemia. The potential collaboration between the UBASH3B/Sts1 variant and CBL mutants could be a subject for future research.
Supplemental References
1.
Mayer RJ, Davis RB, Schiffer CA, et al. Intensive postremission chemotherapy in adults with
acute myeloid leukemia. N Engl J Med. 1994;331(14):896-903.
2.
Moore JO, George SL, Dodge RK, et al. Sequential multiagent chemotherapy is not superior
to high-dose cytarabine alone as postremission intensification therapy for acute myeloid
leukemia in adults under 60 years of age: Cancer and Leukemia Group B study 9222. Blood.
2005;105(9): 3420-3427.
3.
Kolitz JE, George SL, Dodge RK, et al. Dose escalation studies of cytarabine, daunorubicin,
and etoposide with and without multidrug resistance modulation with PSC-833 in untreated
adults with acute myeloid leukemia younger than 60 years: final induction results of Cancer
and Leukemia Group B study 9621. J Clin Oncol. 2004;22(21):4290-4301.
4.
Kolitz JE, George SL, Marcucci G, et al. P-glycoprotein inhibition using valspodar (PSC-833)
does not improve outcomes for patients younger than age 60 years with newly diagnosed
acute myeloid leukemia: Cancer and Leukemia Group B study 19808. Blood.
2010;116(9):1413-1421.
5.
Mikhailik A, Ford B, Keller J, Chen Y, Nassar N, Carpino N. A phosphatase activity of Sts1 contributes to the suppression of TCR signaling. Molecular Cell. 2007; 27(3): 486 – 497.
Supplemental Figure legends
Figure S1 (relates to Figure 1)
A. Bar graphs showing frequency of CD34+ and CD11b+ cells in CB cells transduced with various
CBL constructs or a mutant RUNX1 (S291fs) at 1 or 2 weeks of culture. Same data for week 2 are
shown as FACS plots in Figure 1C. B. Wright-Giemsa-stained cytospins of CB cells expressing
various CBL constructs or a mutant RUNX1 (S291fs) cultured for 3 weeks, showing macrophage
morphology in the wild-type or mutant CBL-transduced cultures. The S291fs-transduced culture
contained cells with an immature myeloid phenotype. C, D. An independent experiment to assess the
frequency of GFP+ cells (vector/CBL/mutant-expressing cells) (C) or the frequency of CD34+ cells at
1 and 3 weeks (D) in a CB cell culture. CBL mutants showed a growth-promoting effect in CB cells at
the early phase of culture. Results are normalized to the frequency of GFP+ cells at day 2, set to 1 (C).
However, all CBL mutants decreased CD34 expression in CB cells compared to vector control, while
a RUNX1 mutant (S291fs) increased it. A bar graph and FACS plots are shown (D).
Figure S2 (relates to Figure 2)
A. Expression of the individual proteins was confirmed in AML1-ETO-expressing CB cells. B.
Changes of GFP+ (vector/CBL/mutant-expressing) cell numbers in AML1-ETO cell culture. CBL
mutants showed a robust growth-promoting effect in AML1-ETO cells. Results are normalized to the
number of GFP+ cells at day 3, set to 1. C. Changes of frequency of GFP+ (vector/CBL/mutantexpressing) cell frequency in 2 additional cultures using AML1-ETO-expressing cord blood cells
derived from different donors. Results are normalized to the frequency of GFP+ cells at day 3, set to 1.
CBL mutants showed a robust growth-promoting effect in AML1-ETO cells.
Figure S3 (relates to Figure 2B)
A. Human CB CD34+ cells were cotransduced with various CBL constructs (marked with GFP)
together with AML1-ETO (marked with Thy1.1), and were cultured with cytokines to monitor the
changes of GFP/Thy1.1 expression. FACS plots of 2 independent experiments are shown. B. Protein
expression of wild-type and mutant CBL in cells at day 45 of culture.
Figure S4 (relates to Figure 2C)
Three independent human CB CD34+ cells were cotransduced with various CBL constructs (marked
with GFP) together with AML1-ETO (marked with Thy1.1), followed by injection of 5x105 cells into
the left femur of NSGS mice. Bone marrow cells from the injected bone were analyzed 12-16 weeks
after injection. FACS plots for Thy1.1 (AML1-ETO) and GFP (vector/CBL/mutants) expression in
human CD45+ cells are shown. There was a strong trend that mutant CBL increases the frequency of
GFP/Thy1.1-DP cells in recipient mice.
Figure S5 (relates to Figure 2C)
A. Wright-Giemsa staining and flow cytometry analyses of AML1-ETO/CBL-mutant coexpressing
cells. The cells were CD33+ myeloid progenitors in almost all cases, except for one case in which
CD19+ lymphoid progenitors were expanded. Both myeloid and lymphoid progenitors contained
immature blast-like cells. B. Surface expression of myeloid/lymphoid markers in the engrafted human
cells expressing AML1-ETO and mutant CBL. Myeloid cells (most cases) are CD33+, CD13+,
CD11b+/-, CD14+/-, and CD19-. Lymphoid cells (detected only in one mouse) are CD19+, CD79a+,
CD34+/-, CD33-, and MPO-. C. The human CD45+ cells expressing mutant CBL (GFP+) were
detected not only in injected bones but also in noninjected bones and spleens. D. Protein expression of
AML1-ETO and mutant CBL in the engrafted human myeloid cells was confirmed by western
blotting.
Figure S6 (relates to Figure 3)
A. CBL was abundantly expressed in human CB cells. CB cells expressing leukemia-fusion proteins
(AML1-ETO, CBFB-MYH11, MLL-AF9), and myeloid leukemia cell lines, as indicated. B. Scheme
of shRNA-resistant version of CBL with silent mutations within the shCBL targeting sequences (shRCBL). E8/9 lacks the sequences targeted by shCBL. TKB: Tyrosine Kinase binding domain, Linker:
Linker domain, RING: RING finger domain, PR: Proline-Rich domain, LZ: leucine zipper motif. C.
CBL constructs and shCBL were cotransfected in 293T cells, and the protein expression was assessed.
shCBL downregulated the expression of wild-type CBL, but did not downregulate those of shR-CBL
and E8/9. D. An independent experiment to that for Figure 3C. AML1-ETO cells were transduced
with shCBL (marked with Venus) in combination with vector or shR-CBL or E8/9 (marked with
GFP), and the growth of Venus+ (shRNA only) and GFP+Venus+ (shRNA + vector or shR-CBL or
E8/9) cells were monitored. Results are normalized to the frequency of Venus+ cells or that of
GFP+Venus+ cells at day 3, set to 1. The growth-promoting effect of shCBL was abrogated by shRCBL. In this clone of AML1-ETO cells, E8/9 mutant showed synergistic effect with shCBL to
promote the growth of AML1-ETO cells.
Figure S7 (relates to Figure 4A)
A, B. AML1-ETO cells were transduced with NT or shCBL (A), or with vector or wild-type/mutant
CBL (B), and were cultured with cytokines. Cell cycle status and apoptosis were assessed on days 5
through 7 of culture. The normalized data are shown in Figure 4A.
Figure S8 (relates to Figure 4C)
The intensities of phosphorylated proteins relative to total STAT5, AKT, ERK and Src, shown in
Figure 4C, are plotted.
Figure S9 (relates to Figure 4C)
A. AML1-ETO-expressing CB cells and Kasumi-1 cells were transduced with NT or shCBL. The
cells were left unstimulated or stimulated with 6 cytokines (SCF, TPO, FLT3L, IL-3, IL-6, GM-CSF)
for the indicated times. Total cell lysates were analyzed by western blotting, using antibodies to
STAT5, AKT, ERK, their phosphorylated forms, and Tubulin. Note the higher level of STAT5
phosphorylation in CBL-depleted AML1-ETO cells at 60 and 120 minutes, and higher level of
AKT/ERK phosphorylation in CBL-depleted Kasumi-1 cells at 30, 60 and 120 minutes. B. The
intensities of phosphorylated proteins relative to total STAT5, AKT and ERK are plotted.
Figure S10 (relates to Figure 4C)
AML1-ETO-expressing CB cells were transduced with vector, CBL, or E8/9 mutant. The cells were
left unstimulated or stimulated with TPO (100 ng/ml) alone for the indicated times. Total cell lysates
were analyzed by western blotting using antibodies to phosphor-STAT5 and Tubulin. The intensities
of phosphorylated STAT5 relative to Tubulin are plotted. Note the sustained signal of phosphorylated
STAT5 in ΔE8/9-expressing cells following TPO stimulation.
Figure S11 (relates to Figure 5A)
(Left) A plot of UBASH3A/Sts-2 expression in human AML samples and human normal
hematopoietic populations was drawn using HemaExplorer. UBASH3A/Sts-2 expression is low in
AML1-ETO (AE) and CBFB-MYH11 (CM) leukemia compared to human CD4+ and CD8+ T cells
and megakaryocytic/erythroid progenitor cells (MEP). Meaning of other abbreviations is available in
the HemaExplorer website (http://servers.binf.ku.dk/hemaexplorer/). (Right) A scatter plot showing
CBL expression against UBASH3A/Sts-2 expression in 187 AML patient samples was drawn using
cBioPortal. Pearson’s correlation= -0.23, Spearman’s correlation= -0.19.
Figure S12 (relates to Figure 6)
A putative miR-9 binding site in UBASH3B/Sts-1 gene is evolutionally conserved. TargetScan
(http://www.targetscan.org/) was used for this analysis.
Figure S13 (relates to Figure 7)
A. AML1-ETO-expressing CB cells transduced with NT, shUBASH-1, or shUBASH-2 were left
unstimulated or stimulated with 6 cytokines for 5 minutes. Total cell lysates were analyzed by western
blotting using the indicated antibodies. B. AML1-ETO- and CBL mutant-expressing CB cells
transduced with NT or shUBASH-1 were left unstimulated or stimulated with 6 cytokines for 5
minutes. Total cell lysates were analyzed by western blotting using the indicated antibodies. C.
AML1-ETO cells were transduced with shUBASH-1 or shUBASH-2 (marked with Venus) in
combination with vector, wild-type CBL or E8/9 mutant (marked with GFP), and the growth of
Venus+ (shRNA only) and GFP+Venus+ [shRNA + (vector or CBL or E8/9)] cells were
monitored. Results are normalized to the frequency of Venus+ cells or that of GFP+Venus+ cells at
day 3, set to 1. E8/9 reversed the negative effect of shUBASH at least partially, while wild-type
CBL did not.
Figure S14
Changes of Venus+ cells (shRNA-transduced cells) in the culture of CBF-MYH11- or MLL-AF9expressing CB cells. Results are normalized to the frequency of GFP+ cells at day 2, set to 1. shCBL
showed growth-promoting or inhibitory effect compared to non-targeting (NT) control in CBFBMYH11 or MLL-AF9 cells, respectively.