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Supplemental material
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“KRasG12D-evoked leukemogenesis does not require -catenin” by Cherry Ee Lin Ng, Amit Sinha, Andrei
Krivtsov, Stuart Dias, Jenny Chang, Scott A. Armstrong and Demetrios Kalaitzidis
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Material and methods
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Mice
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LSL-KRasG12D, Mx1-Cre, and floxed -catenin mice described previously were intercrossed,1,2 and
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recipients and source of competitor/helper bone marrow (BM) (Taconic, Hudson, NY, USA). 15 g/g body
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USA). CT values were calculated for each primer set by first normalizing to an endogenous control within
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TTG GTG A). CT was then calculated by normalizing CT values to those of known positive controls
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(CT flanking + CT excised)) x 100%.
maintained in the C57Bl/6 background (all CD45.2). B6.SJL (CD45.1) mice were used as transplant
weight of polyinosinic-polycytidylic acid (pIpC) (GE Healthcare Lifesciences, Pittsburgh, PA, USA),
dissolved in PBS, was injected into mice in the peritonium when indicated. All mice were housed under
specific pathogen-free conditions in an accredited facility at Children’s Hospital Boston. All experiments
were conducted after approval by the Institutional Animal Care and Use Committee (IACUC).
PCR
Genotyping PCR for maintenance of mice was carried out as described previously. 1,2 For detection of βcatenin excision, primers flanking the excised region (mβcat-del-qPCR-FW1: CAA ATC TTT GAG CCT
GTG TGT G; mβcat-del-qPCR-RV1: GGC TAT TGG GAT TTC CAG GTA T) as well as primers within the
excised regions (mβcat-deleted-qPCR-FW1: TCA ACA TCT GTG ATG GTT CAG C; mβcat-deletedqPCR-RV1: ATG TTC CCT GAG ACG CTA GAT G) were used in quantitative real-time PCR (qRT-PCR)
reactions. qRT-PCR reactions were run on Applied Biosystems 7500 RT PCR Systems (Carlsbad, CA,
the β-catenin gene locus that is common to both excised and non-excised templates (mβcat-gDNAqPCR-FW1: ACT GGT ATG GAA GTC CCT GTG A; mβcat-gDNA-qPCR-RV1: GCT CCC ATT TTA TCT
(i.e. template known to have complete β-catenin excision when using primers flanking the excised region
and template known to have an intact β-catenin locus when using primers within the excised regions). To
determine the percent excision of β-catenin alleles, the following formula is applied: (CT flanking /
Western blots
Whole cell lysates were obtained by boiling cells in lysis buffer (1% SDS, 1.0mM sodium ortho-vanadate,
10mM Tris pH 7.8) before passing through a 28-gauge needle. Lysates were then centrifuged for 15 min
and supernatant was retained for protein concentration determination using the BCA protein assay
(Pierce, Rockford, IL, USA). 50-100 µg of protein were loaded per sample for western-blot analyses. The
antibodies used were anti-β-Catenin (clone 14) [BD Transduction Laboratories, San Jose, CA, USA] and
anti-Lamin B1 (ab16048) [Abcam, Cambridge, MA, USA].
T-ALL experiments
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CD45.1 recipient mice received lethal doses of irradiation (two doses at 450 rad, 3 hr apart) prior to
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-catenin excision by qRT-PCR.
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Catloxp/loxpKrasG12DMxCre+,
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Catloxp/loxpMxCre+ (i.e. Cat-/- upon pIpC induced excision of floxed alleles in transplant recipients). Sorted
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from moribund or dead mice. Genomic DNA from erythrocyte-lysed spleen cells was used to check for -
transplantation of 1 x 106 whole bone marrow cells (WBM) from 4-5 week old CD45.2 mice
(Cat+/+KrasG12D, Cat+/-KrasG12D, Cat-/-KrasG12D and Catlox/loxp) together with an equal number of CD45.1
WBM cells by retro-orbital injection on the same day. Donor cell chimerism was assessed by flow
cytometric analysis of CD45.2 cells in the peripheral blood of recipient mice starting at 4 weeks after
transplantation. Complete blood counts were performed on PB samples using a Hemavet (Drew
Scientific, Dallas, TX). To ensure complete excision of floxed alleles, pIpC was administered by intraperitoneal injections three-times over 5-6 days, from 6 weeks after transplantation. Three mice from each
group were sacrificed at 15 weeks 5 days after transplantation for further analyses. Thymus cells from
these mice were frozen down and stored in liquid nitrogen until their use for secondary limiting-dilution
transplantation into sub-lethally irradiated (600 rad) CD45.1 recipient mice. When moribund, mice were
euthanized and subject to analyses. Genomic DNA from erythrocyte-lysed BM cells was used to assess
AML experiments
CD45.2 recipient mice received sub-lethal doses of irradiation (single dose of 600 rad) prior to
transplantation of MLL-AF9 transduced Lineage-Sca-1+c-Kit+ (LSK)-derived BM cells on the same day.
Briefly, BM LSK cells were sorted from donor mice of the following genotype: Cat+/+KrasG12DMxCre+,
Catloxp/loxpKrasG12D-LSLMxCre-
(i.e.
Catloxp/loxpMLL-AF9)
and
LSK cells were transduced on the same day with MSCV-MLL-AF9-ires-GFP retrovirus. Two days after
viral transduction, cells were transferred into Methocult®M3234 (Stemcell Technologies Vancouver, BC,
Canada) supplemented with IL-3, IL-6, SCF, TPO and Flt-3 and remained in semi-solid culture for five
days prior to collection. Viable GFP+ cells were sorted and transplanted into recipient mice by retro-orbital
injection on the same day at a dose of 3 x 103 GFP+ cells per recipient mouse. Excision of floxed alleles
only in MLL-AF9 transplants (not KRasG12DMLL-AF9 transplants) was induced by intra-peritoneal
injections of pIpC three-times over 5-6 days, from 6 weeks after transplantation. Kras G12DMLL-AF9
recipient mice were euthanized and analyzed at 4 weeks after transplantation while MLL-AF9 recipients
were euthanized and analyzed at 8 weeks 3 days after transplantation. Erythrocyte-lysed BM and spleen
cells were harvested from these mice and were stored frozen in liquid nitrogen until use for secondary
limiting dilution transplantation. For secondary limiting dilution transplantation, BM cells were transplanted
at various concentrations (1 x 104, 1x 103 and 1 x 102) into sub-lethally irradiated (600 rad) CD45.1
recipient mice. A single dose of pIpC was administered by intra-peritoneal injection at 7-10 days after
transplantation to ensure complete excision of floxed alleles. Spleen, thymus and/or BM were harvested
catenin excision by qRT-PCR.
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For all limiting-dilution transplantation assays, frequency of LICs was analyzed and determined by L-
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References
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1.
Heidel FH, Bullinger L, Feng Z, Wang Z, Neff TA, Stein L, et al. Genetic and
pharmacologic inhibition of beta-catenin targets imatinib-resistant leukemia stem
cells in CML. Cell Stem Cell 2012 Apr 6; 10(4): 412-424.
2.
Chan IT, Kutok JL, Williams IR, Cohen S, Kelly L, Shigematsu H, et al.
Conditional expression of oncogenic K-ras from its endogenous promoter
induces a myeloproliferative disease. J Clin Invest 2004 Feb; 113(4): 528-538.3.
Calc™ (Stemcell Technologies).
Flow Cytometry
BM cells were harvested and processed as previously described. 3 As part of leukemia assessment of
mice, cells were stained, in various combinations as required, with antibodies (clones) against the
following: CD3 (17A2), CD4 (RM4-5), CD8a (53-6.7), CD19 (6D5), B220 (RA3-6B2), Gr-1 (RB6-8C5),
CD127 (A7R34) and Ter-119 (TER-119), CD117 (2B8), CD11b (M1/70), Sca-1 (D7 or E13-161.7), CD150
(SLAM), CD16/32 (93), CD25 (PC61), CD44 (IM7), CD34 (RAM34), Flt-3 (A2F10), CD26 (H194-112)
(Biolegend, San Diego, CA, USA) and CD99 (R&D systems Minneapolis, MN, USA); and acquired on a
BD LSR II. Data were analyzed using FlowJo™ (Tree Star, Ashland, OR, USA). BM cells used in sorting
experiments were enriched for Lin- cells by staining with a cocktail of biotin-conjugated antibodies against
CD3 (17A2), CD4 (RM4-5), CD8a (53-6.7), CD19 (6D5), B220 (RA3-6B2), Gr-1 (RB6-8C5), IL-7R
(A7R34) and Ter-119 (TER-119), followed by incubation with streptavidin-conjugated Dynabeads
(Invitrogen, Carlsbad, CA, USA) and undergoing cell separation with magnetic columns. Anti-mouse
CD117 (2B8) was used in combination to sort out Lin-c-KithiGFP+ BM cells.
Gene expression analysis
For microarray experiments, 4-6 x 105 Lin-KithiGFP+ cells were sorted from each of the leukemic mice,
RNA was isolated with Trizol and hybridized to Affymetrix murine 430 2.0 arrays. Expression data were
analyzed with GenePattern release 3.1 software package (http://www.broad.mit.edu/tools/software.html).
Microarray data have been deposited at the NCBI Gene Expression Omnibus with accession code
GSE49248.
Statistical Analysis
The statistical significance of differences between population means was assessed by two-tailed unpaired
Student’s t test unless otherwise indicated.
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3.
Kalaitzidis D, Sykes SM, Wang Z, Punt N, Tang Y, Ragu C, et al. mTOR complex
1 plays critical roles in hematopoiesis and Pten-loss-evoked leukemogenesis.
Cell Stem Cell 2012 Sep 7; 11(3): 429-439.
Tables
WBC
(x 103 per ml)
Spleen weight
(mg)
Liver weight
(mg)
Thymus weight
(mg)
8.1  1.2
n=11
94.8 14.2
n=6
856 48.8
n=6
60.2  15.0
n=6
βCat+/+KrasG12D
36.7  13.4
n=9
969.5 125.7
n=6
1698.0 ± 229.1
155.3  48.0
n=6
βCat+/-KrasG12D
41.3  14.2
n=7
972.3  118.1
n=7
1746.9 ± 238.8
41.8  19.5
n=5
1110.0  156.6
n=5
1757.6 ± 261.3
n=5
197.2  113.3
n=5
T-ALL (primary)
βCatloxp/loxp
9.0  0.5
96.7  7.2
N.D.
81.3  12.8
n=8
n=3
βCat+/+KrasG12D
10.8  1.6
397.1  31.3
N.D.
366.3  55.1
MPD (JMML/CMML)
βCatloxp/loxp
βCat-/-KrasG12D
n=6
n=7
301.1  94.3
n=7
n=3
n=7
n=7
βCat+/-KrasG12D
9.0  1.3
403.0  53.2
n=6
n=4
βCat-/-KrasG12D
10.5  1.1
395.0  40.3
n=5
n=4
72.7  41.7
273.2  36.0
1231.1  152.3
39.2  5.9
n=3
n=7
n=7
n=7
15.5  4.0
251.7  58.0
945.0  124.2
77.1  30.6
n=3
n=7
n=7
n=7
231.3  8.7
n=3
702.0  41.4
n=3
2471.7  50.0
n=3
60.7  9.5
n=3
βCat-/-KrasG12DMLL-AF9
249.8  33.7
n=2
852.0  107.0
n=2
3077.5  188.5
n=2
62.0  27.0
n=2
βCatloxp/loxpMLL-AF9
262.6  118.1
n=3
665.7  128.4
n=3
2048.7  171.6
n=3
40.7  9.3
n=3
68.4  53.1
n=3
757.8  42.6
n=4
2349.0  266.3
n=4
35.0  7.5
n=3
T-ALL (secondary)
βCat+/+KrasG12D
βCat-/-KrasG12D
AML (primary)
βCat+/+KrasG12DMLL-AF9
βCat-/-MLL-AF9
n=7
N.D.
349.0  81.2
N.D.
344.0  78.0
n=4
n=5
AML (secondary)
βCat+/+KrasG12DMLL-AF9
βCat-/-KrasG12DMLL-AF9
βCatloxp/loxpMLL-AF9
382.6  61.7
n=3
544.1 35.6
n=13
2195.7  155.1
n=13
40.9  2.8
n=13
231.3  35.4
n=3
464.6  40.5
n=11
1587.9 ± 146.2
40.5 ± 6.1
n=11
n=11
218.5  63.4
n=4
424.5 ± 45.2
n=11
1734.8  130.2
n=11
37.7  6.6
n=11
85.4  58.9
391.4  71.9
1406.6  144.6
60.5 7.9
n=10
n=10
βCat-/-MLL-AF9
n=5
n=10
Abbreviations: WBC, white blood cell; N.D., not determined
Table S1. Analyses of leukemic mice
βCat+/+KrasG12D
βCat-/-KrasG12D
number of dead mice / total number of
mice transplanted
3/4
6/6
No. of cells transplanted
1 x 106
1 x 105
3/5
3/6
104
1/6
0/6
1 in 316,103
1 in 160,853
1x
Frequency of LIC
Table S2a. Frequency of Leukemia-Initiating Cells (LIC) in T-ALL
βCat+/+KrasG12D
MLL-AF9
No. of cells
transplanted
1 x 104
βCat+/+MLLAF9
βCat-/-MLL-AF9
number of dead mice / total number of mice transplanted
5/5
5/5
5/5
5/5
1x
103
5/5
4/5
4/5
2/5
1x
102
1/5
2/4
1/5
0/4
1 in 288
1 in 431
1 in 556
1 in 1,992
Frequency of LIC
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βCat-/-KrasG12D
MLL-AF9
Table S2b. Frequency of Leukemia-Initiating Cells (LIC) in AML
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Figure Legends
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Figure S1.
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Figure S2.
β-catenin is dispensable for KRasG12D-induced MPN (a) Comparison of
average spleen weights illustrates splenomegaly in all mice expressing oncogenic
KRas, regardless of β-catenin status. (b) Increased percentages of myeloid cells
(Mac1+Gr1lo/neg and Mac+Gr1+) in the spleen of mice expressing KRasG12D, regardless of
β-catenin status. Numbers of peripheral blood (c) white blood cells (WBC) and (d)
neutrophils (NE) are increased in mice expressing KRasG12D, regardless of β-catenin
status. Error bars indicate the standard error of the mean (s.e.m.). Abbreviations: N.S. =
not significant; * = p<0.05
β-catenin is dispensable for KRasG12D-induced T-ALL. (a) Western blot (WB)
analysis using lysates from thymocytes of primary transplant recipient mice showing
deletion of full length β-catenin and the presence of truncated β-catenin in βCat-/KRasG12D thymocytes (upper panel); Lamin B1 is used as loading control (lower panel).
Molecular weights (kDa) are shown on the right. (b) Quantitative real-time PCR was
performed to assess β-catenin excision in thymocytes [as in (a)] as well as cells from
the BM and spleens of all recipient mice transplanted with βCat-/-KRasG12D cells.
Primers flanking excised regions allows for amplification of β-catenin excision products
while primers within excised regions allow for the amplification of wild-type, non-excised
β-catenin. For βCat+/+KRasG12D and βCat+/-KRasG12D thymocytes (thy), error bars show
the standard deviation (s.d.); for all other βCat-/-KRasG12D data shown, error bars show
s.e.m. Abbreviations: Thy = thymocytes; BM = bone marrow; spl = spleen. (c)
Thymocytes from 3 mice per group of recipient mice were analyzed by flow cytometry
showing that mice transplanted with BM cells expressing KRasG12D, even in the absence
of β-catenin, exhibit abnormal CD4/CD8 profiles (center and right panels), clearly
distinct from that of normal thymocytes (left panel). Percentage of total thymocytes is
shown in each quadrant.
Figure S3. Loss of β-catenin does not affect the frequency of leukemia-initiating cells
(LICs) in KRasG12D-induced T-ALL. (a) Quantitative real-time PCR was carried out to
verify excision of β-catenin in BM cells of moribund secondary recipient mice from
Figure 1e. (b) Chimerism of donor cells assessed by flow-cytometric analysis in
thymocytes (Thy) and nucleated cells from the whole BM (WBM), spleen (Spl) and
peripheral blood (PB) of moribund mice, showing no statistical difference in chimerism
between βCat+/+KRasG12D and βCat-/-KRasG12D cells. For WBM, Spl and Thy,
βCat+/+KRasG12D (n=6), βCat-/-KRasG12D (n=6) ; for PB, βCat+/+KRasG12D (n=4), βCat-/KRasG12D (n=5); (c) Flow cytometric analysis of cells from the BM, spleen and thymus
reveal that moribund recipient mice exhibited abnormal CD4/CD8 profiles, reminiscent
of their corresponding donor (1) thymocytes that were used for secondary (2)
transplant. T-ALL (1): Numbers in each quadrant represent percentages. T-ALL (2):
Numbers in each quandrant represent percentages shown as “mean ± s.e.m.” (n=3);
only mean percentages greater than 2 are shown. Abbreviation: n.s., not significant
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Figure S4. β-catenin is dispensable for KRasG12D-MLL-AF9 acute myeloid leukemia
(AML). (a-d) βCat+/+KRasG12DMLL-AF9, n=3; βCat-/-KRasG12DMLL-AF9, n=2;
βCatloxp/loxpMLL-AF9, n=3; βCat-/-MLL-AF9, n=3 (a) WB analysis using lysates from
WBM cells of primary transplant mice shows the loss of full-length β-catenin and a
reciprocal gain of truncated β-catenin in βCat-/-KRasG12DMLL-AF9 and βCat-/-MLL-AF9
cells, confirming Cre-mediated excision within the β-catenin gene locus (upper panel);
Lamin B1 is used as loading control (lower panel). Molecular weights (kDa) are shown
on the right. (b) Chimerism of donor cells in the peripheral blood is shown, expressed as
percentage of GFP+ cells. (c) Chimerism of donor cells in the spleen is shown,
expressed as percentage of GFP+ cells. (d) Graphical representation of the percentages
of B220+ B cells, CD3+ T cells and Mac1+ myeloid cells in the spleens of recipient mice
is shown. Abbreviations: N.S. = not significant (p>0.05); * = p<0.05. Error bars indicate
s.e.m.
Figure S5. KRasG12D partially compensates for the loss of β-catenin in MLL-AF9
acute myeloid leukemia (AML). (a) Quantitative real-time PCR was carried out to verify
excision of β-catenin in spleen cells of moribund βCat-/-KRasG12DMLL-AF9 and βCat-/MLL-AF9 secondary transplant recipient mice, as in Figure 2c. (b) Flow cytometric
analyses of BM cells obtained from the same mice that cells were used for gene
expression microarray analyses, as in Figure 2d, was performed. Representative
histograms are shown (left) and average mean fluorescence intensity (MFI) is
represented graphically (right); βCat-/-MLL-AF9 (n=5) and all other groups (n=3). CD99
and DPPIV/CD26 are decreased and increased, respectively, on Lin -Sca-kit+GFP+ cells
with the loss of -catenin in MLL-AF9 AML cells. Abbreviations: * = p<0.05. Error bars
indicate s.e.m. unless otherwise stated.