Supplemental material

“KRasG12D-evoked leukemogenesis does not require b-catenin” by Cherry Ee Lin Ng, Amit Sinha, Andrei Krivtsov, Stuart Dias, Jenny Chang, Scott A. Armstrong and Demetrios Kalaitzidis

Material and methods

Mice

LSL-KRasG12D, Mx1-Cre, and floxed b-catenin mice described previously were intercrossed,1,2 and maintained in the C57Bl/6 background (all CD45.2). B6.SJL (CD45.1) mice were used as transplant recipients and source of competitor/helper bone marrow (BM) (Taconic, Hudson, NY, USA). 15 mg/g body 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 β-catenin 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-deleted-qPCR-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, USA). DCT values were calculated for each primer set by first normalizing to an endogenous control within the β-catenin gene locus that is common to both excised and non-excised templates (mβcat-gDNA-qPCR-FW1: ACT GGT ATG GAA GTC CCT GTG A; mβcat-gDNA-qPCR-RV1: GCT CCC ATT TTA TCT TTG GTG A). DDCT was then calculated by normalizing DCT values to those of known positive controls (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: (DDCT flanking / (DDCT flanking + DDCT excised)) x 100%.

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

CD45.1 recipient mice received lethal doses of irradiation (two doses at 450 rad, 3 hr apart) prior to transplantation of 1 x 106 whole bone marrow cells (WBM) from 4-5 week old CD45.2 mice (bCat+/+KrasG12D, bCat+/-KrasG12D, bCat-/-KrasG12D and bCatlox/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 intra-peritoneal 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 b-catenin excision by qRT-PCR.

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: bCat+/+KrasG12DMxCre+, bCatloxp/loxpKrasG12DMxCre+, bCatloxp/loxpKrasG12D-LSLMxCre- (i.e. bCatloxp/loxpMLL-AF9) and bCatloxp/loxpMxCre+ (i.e. bCat-/- upon pIpC induced excision of floxed alleles in transplant recipients). Sorted 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. KrasG12DMLL-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 from moribund or dead mice. Genomic DNA from erythrocyte-lysed spleen cells was used to check for b-catenin excision by qRT-PCR.

For all limiting-dilution transplantation assays, frequency of LICs was analyzed and determined by L-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.

References

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.

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)
MPD (JMML/CMML)
βCatloxp/loxp / 8.1 ± 1.2 / 94.8 ± 14.2 / 856 ± 48.8 / 60.2 ± 15.0
n=11 / n=6 / n=6 / n=6
βCat+/+KrasG12D / 36.7 ± 13.4 / 969.5 ± 125.7 / 1698.0 ± 229.1 / 155.3 ± 48.0
n=9 / n=6 / n=6 / n=6
βCat+/-KrasG12D / 41.3 ± 14.2 / 972.3 ± 118.1 / 1746.9 ± 238.8 / 301.1 ± 94.3
n=7 / n=7 / n=7 / n=7
βCat-/-KrasG12D / 41.8 ± 19.5 / 1110.0 ± 156.6 / 1757.6 ± 261.3 / 197.2 ± 113.3
n=5 / n=5 / n=5 / 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 / n=3
βCat+/+KrasG12D / 10.8 ± 1.6 / 397.1 ± 31.3 / N.D. / 366.3 ± 55.1
n=7 / n=7 / n=7
βCat+/-KrasG12D / 9.0 ± 1.3 / 403.0 ± 53.2 / N.D. / 349.0 ± 81.2
n=6 / n=4 / n=4
βCat-/-KrasG12D / 10.5 ± 1.1 / 395.0 ± 40.3 / N.D. / 344.0 ± 78.0
n=5 / n=4 / n=5
T-ALL (secondary)
βCat+/+KrasG12D / 72.7 ± 41.7 / 273.2 ± 36.0 / 1231.1 ± 152.3 / 39.2 ± 5.9
n=3 / n=7 / n=7 / n=7
βCat-/-KrasG12D / 15.5 ± 4.0 / 251.7 ± 58.0 / 945.0 ± 124.2 / 77.1 ± 30.6
n=3 / n=7 / n=7 / n=7
AML (primary)
βCat+/+KrasG12DMLL-AF9 / 231.3 ± 8.7 / 702.0 ± 41.4 / 2471.7 ± 50.0 / 60.7 ± 9.5
n=3 / n=3 / n=3 / n=3
βCat-/-KrasG12DMLL-AF9 / 249.8 ± 33.7 / 852.0 ± 107.0 / 3077.5 ± 188.5 / 62.0 ± 27.0
n=2 / n=2 / n=2 / n=2
βCatloxp/loxpMLL-AF9 / 262.6 ± 118.1 / 665.7 ± 128.4 / 2048.7 ± 171.6 / 40.7 ± 9.3
n=3 / n=3 / n=3 / n=3
βCat-/-MLL-AF9 / 68.4 ± 53.1 / 757.8 ± 42.6 / 2349.0 ± 266.3 / 35.0 ± 7.5
n=3 / n=4 / n=4 / n=3
AML (secondary)
βCat+/+KrasG12DMLL-AF9 / 382.6 ± 61.7 / 544.1 ± 35.6 / 2195.7 ± 155.1 / 40.9 ± 2.8
n=3 / n=13 / n=13 / n=13
βCat-/-KrasG12DMLL-AF9 / 231.3 ± 35.4 / 464.6 ± 40.5 / 1587.9 ± 146.2 / 40.5 ± 6.1
n=3 / n=11 / n=11 / n=11
βCatloxp/loxpMLL-AF9 / 218.5 ± 63.4 / 424.5 ± 45.2 / 1734.8 ± 130.2 / 37.7 ± 6.6
n=4 / n=11 / n=11 / n=11
βCat-/-MLL-AF9 / 85.4 ± 58.9 / 391.4 ± 71.9 / 1406.6 ± 144.6 / 60.5 ± 7.9
n=5 / n=10 / n=10 / n=10
Abbreviations: WBC, white blood cell; N.D., not determined
Table S1. Analyses of leukemic mice
βCat+/+KrasG12D / βCat-/-KrasG12D
No. of cells transplanted / number of dead mice / total number of mice transplanted
1 x 106 / 3/4 / 6/6
1 x 105 / 3/5 / 3/6
1 x 104 / 1/6 / 0/6
Frequency of LIC / 1 in 316,103 / 1 in 160,853
Table S2a. Frequency of Leukemia-Initiating Cells (LIC) in T-ALL
βCat+/+KrasG12D MLL-AF9 / βCat-/-KrasG12D MLL-AF9 / βCat+/+MLL-AF9 / βCat-/-MLL-AF9
No. of cells transplanted / number of dead mice / total number of mice transplanted
1 x 104 / 5/5 / 5/5 / 5/5 / 5/5
1 x 103 / 5/5 / 4/5 / 4/5 / 2/5
1 x 102 / 1/5 / 2/4 / 1/5 / 0/4
Frequency of LIC / 1 in 288 / 1 in 431 / 1 in 556 / 1 in 1,992

Table S2b. Frequency of Leukemia-Initiating Cells (LIC) in AML

Figure Legends

Figure S1. 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

Figure S2. 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