Cbfb Deﬁciency Results in Differentiation Blocks and Stem/ Progenitor Cell Expansion in Hematopoiesis

Letters to the Editor 753 4 Perez-Persona E, Vidriales MB, Mateo G, Garcia-Sanz R, Mateos MV, de Coca AG 10 Rajkumar SV, Gupta V, Fonseca R, Dispenzieri A, Gonsalves WI, Larson D et al. et al. New criteria to identify risk of progression in monoclonal gammopathy of Impact of primary molecular cytogenetic abnormalities and risk of progression in uncertain significance and smoldering multiple myeloma based on multi- smoldering multiple myeloma. Leukemia 2013; 27: 1738–1744. parameter flow cytometry analysis of bone marrow plasma cells. Blood 2007; 110: 11 Dhodapkar MV, Sexton R, Waheed S, Usmani S, Papanikolaou X, Nair B et al. Clinical, 2586–2592. genomic, and imaging predictors of myeloma progression from asymptomatic 5 Dispenzieri A, Kyle RA, Katzmann JA, Therneau TM, Larson D, Benson J et al. monoclonal gammopathies (SWOG S0120). Blood 2014; 123:78–85. Immunoglobulin free light chain ratio is an independent risk factor for progres- 12 Cherry BM, Korde N, Kwok M, Manasanch EE, Bhutani M, Mulquin M et al. 111 – sion of smoldering (asymptomatic) multiple myeloma. Blood 2008; :785 789. Modeling progression risk for smoldering multiple myeloma: results from 6 Mateos M-V, Hernández M-T, Giraldo P, la Rubia de J, de Arriba F, López Corral L a prospective clinical study. Leuk Lymphoma 2013; 54: 2215–2218. et al. Lenalidomide plus dexamethasone for high-risk smoldering multiple mye- 13 Rajkumar SV, Larson D, Kyle RA. Diagnosis of smoldering multiple myeloma. loma. N Engl J Med 2013; 369: 438–447. N Engl J Med 2011; 365:474–475. 7 Dispenzieri A, Stewart AK, Chanan-Khan A, Rajkumar SV, Kyle RA, Fonseca R et al. 14 Greipp PR. International Staging System for Multiple Myeloma. J Clin Oncol 2005; Smoldering multiple myeloma requiring treatment: time for a new definition? 23 – Blood 2013; 122: 4172–4181. : 3412 3420. 8 Larsen JT, Kumar SK, Dispenzieri A, Kyle RA, Katzmann JA, Rajkumar SV. Serum 15 Bologa RM, Levine DM, Parker TS, Cheigh JS, Serur D, Stenzel KH et al. Interleukin- free light chain ratio as a biomarker for high-risk smoldering multiple myeloma. 6 predicts hypoalbuminemia, hypocholesterolemia, and mortality in hemodialysis 32 – Leukemia 2012; 27: 941–946. patients. Am J Kidney Dis 1998; : 107 114. 9 Neben K, Jauch A, Hielscher T, Hillengass J, Lehners N, Seckinger A et al. 16 Kastritis E, Terpos E, Moulopoulos L, Spyropoulou-Vlachou M, Kanellias N, Progression in smoldering myeloma is independently determined by the Eleftherakis-Papaiakovou E et al. Extensive bone marrow infiltration and abnormal chromosomal abnormalities del(17p), t(4;14), gain 1q, hyperdiploidy, and freelight chain ratio identifies patients with asymptomatic myelomaat high risk tumor load. J Clin Oncol 2013; 31: 4325–4332. for progression to symptomatic disease. Leukemia 2012; 27: 947–953.

Cbfb deﬁciency results in differentiation blocks and stem/ progenitor cell expansion in hematopoiesis

Leukemia (2015) 29, 753–757; doi:10.1038/leu.2014.316 mice where distinction between the two mouse models is not necessary. Deletion of the Cbfb locus was nearly complete in the bone marrow (BM) of Cbfb cKO mice in both mouse models The PEBP2/CBF heterodimeric transcription factors consist (Supplementary Figure 1A and 1B). of two subunits: the DNA-binding α subunit and the non-DNA- In initial hematological analysis, Cbfb cKO mice revealed binding β subunit. The β subunit, Cbfβ, encoded by the Cbfb gene, decreases in all three blood parameters, namely leukocyte, serves to increase the DNA binding ability of the α subunit in an hemoglobin and platelet counts (Figure 1a). Although not allosteric manner and protects it from protein degradation. apparent at early time points, Cbfbfl/fl;Vav-iCre+ mice progressively The α subunit is encoded by three distinct Runx genes: Runx1, showed more drastic decreases in the blood counts. Probably due Runx2 and Runx3. Of these, Runx1 is well established as an to this pronounced pancytopenia, Cbfbfl/fl;Vav-iCre+ mice died by important regulator of hematopoiesis. Consistent with Cbfβ being 6 months old. Lethality was also observed in Cbfbfl/fl;Mx1-Cre+ mice − / − indispensible for Runx1 function, Cbfb murine embryos showed at later time points with much lower frequency (Figure 1b). As − / − the same spectrum of abnormalities found in Runx1 embryos: moribund Cbfbfl/fl;Mx1-Cre+ mice did not exhibit further drop in − / − Cbfb embryos die at embryonic day (E) 12.5 due to blood counts, they are thought to succumb to fatal bacterial hemorrhage in the central nervous system accompanied by the 1 infection. inability to generate hematopoietic stem cells (HSCs). At the adult Subsequent flow-cytometric and morphological analyses stage, Runx1 conditional knockout (cKO) mice led to an expanded showed that the pancytopenia in Cbfb cKO mice was caused by 2,3 HSC compartment and subsequent stem cell exhaustion. differentiation blocks in all hematopoietic lineages, including the In addition, Runx1 cKO mice show differentiation blocks in hi hi 3,4 reduction of mature Mac1 Gr1 granulocytes, accompanied by an megakaryocyte and lymphocyte lineages. Due to the embryonic hi int − / − increase in the Mac1 Gr1 immature granulocyte precursor lethality of Cbfb mice, Cbfb function in adult hematopoiesis has population (Figures 1c and d; Supplementary Figure 2A), and a not been investigated. decrease in B220+CD19+ B cells due to a significant block in RUNX1 and CBFB are frequent targets of mutations in human − maturation from B220intIgD to B220hiIgD+ B cells (Figure 1e; leukemia. Approximately 30% of acute leukemia carry RUNX1 Supplementary Figure 2B). T-cell development was abrogated at genetic alterations, such as chromosomal translocations and point 5–7 double-negative 1 (DN1) to DN2 stage, resulting in drastic mutations, resulting in loss of RUNX1 activity. CBFB is altered in – an inversion of chromosome 16, inv(16)(p13q22), or the less reduction in thymus weight (Figures 1f h; Supplementary Figure 2C). The block in erythroid lineage was exhibited common t(16;16)(p13;q22), generating the CBFB-MYH11 fusion hi 8 by a decrease in Ter119 erythroblasts with an increase in gene associated with the M4Eo subtype of AML. Cbfb-MYH11 int hi knock-in (KI) mice developed phenotypes that are indistinguish- Ter119 CD71 pro-erythroblasts in the BM and the spleen able from Cbfb− / − or Runx1− / − mice,9 indicating the fusion gene (Figure 1i; Supplementary Figure 2D and data not shown). product, CBFβ-SMMHC protein, acts as a dominant repressor of Consistent with severe thrombocytopenia, mature megakaryo- PEBP2/CBF function. cytes with polynuclei were not found in histological analysis (data To examine the functions of Cbfb in adult hematopoiesis, we not shown). Instead, an increase in CD41+CD61+ megakaryocytic analyzed Cbfb conditional knockout (cKO) mice utilizing the Mx1- lineage cells was observed (Figures 1j and k; Supplementary Cre and Vav-iCre systems. Cbfbfl/fl;Mx1-Cre+ and Cbfbfl/fl;Vav-iCre+ Figure 2E), perhaps as an accumulation of megakaryoblasts due to mice were generated and are collectively referred to as Cbfb cKO the block in megakaryocyte differentiation.

Accepted article preview online 5 November 2014; advance online publication 25 November 2014

* ** 15 16 * *** 15 *** *** *** *** /µL)

3 12 /µL)

10 3 10 8 5 5 4 Platelets (10 Hemoglobin (g/dL) Leukocytes (10 0 0 0 Cbfbf/f Cbfbf/f; Cbfbf/f Cbfbf/f; Cbfbf/f Cbfbf/f; Cbfbf/f Cbfbf/f; Cbfbf/f Cbfbf/f; Cbfbf/f Cbfbf/f; Mx1-Cre Vav-iCre Mx1-Cre Vav-iCre Mx1-Cre Vav-iCre

Cbfbf/f 100 60 * Cbfbf/f;Mx1-Cre *** Cbfbf/f 80 Cbfbf/f;Vav-iCre ** *** 40 60

** 40 20 f/f Percent survival (Cbfb vs f/f

20 Cbfb ;Vav-iCre) Frequency in BM (%) p = 0.0075 0 0 100 200 300 400 Mac1hiGr1hi Mac1hiGr1int Age (days)

BM * ** 20 25 ** *** *** *** 100

cells (%) 20 Cbfbf/f 15 * - 15 *** 10 50 10 5 Cbfbf/f; 5

Vav-iCre Frequency in BM (%)

0 0 Thymus weight (mg) 0 Frequency in CD43 Cbfbf/f Cbfbf/f; Cbfbf/f Cbfbf/f; B220+CD19+ B220hiIgD+ Mx1-Cre Vav-iCre

100 100

50 20 * *** cells (%) 50 - 10 CD8 * -

Frequency in ** CD4

Frequency in thymus (%) 0 0 + CD4-CD8- CD4+CD8+ CD4+CD8- CD4-CD8 DN1 DN2 DN3 DN4

* 60 100 50 * 25 ** *** *

cells (%) 80 40 20

hi * 40 60 * 30 15

40 20 10 20 * 20 10 5 Frequency in BM (%) *** ** Frequency in BM (%) Frequency in spleen (%)

0 Ter119 Frequency in 0 0 0 Ter119 int Ter119 hi Ter119 hi Ter119hi Ter119hi CD41+CD61+ CD41+CD61+ CD71hi CD71hi CD71hi CD71lo FSChi FSClo FSClo

Leukemia (2015) 739 – 757 © 2015 Macmillan Publishers Limited Letters to the Editor 755 To evaluate whether the pancytopenia could also be due Cbfb cKO mice showed more pronounced differentiation to a defect at the stem cell level, we examined the immuno- blockages and HSPC expansion than Runx1 cKO mice fl fl phenotypically defined c-Kit+Sca-1+Lineage− (KSL) population (Supplementary Figure 4A). Interestingly, Cbfb / ;Vav-iCre+ mice which is enriched for hematopoietic stem cells (HSCs). Surprisingly, are not born at Mendelian ratios (Supplementary Table 1), unlike fl fl the KSL population exhibited a significant increase in the BM of Runx1 / ;Vav-iCre+ mice.10 These results suggest the existence of Cbfb cKO mice (Figure 2a), which is mainly due to the drastic functional compensation mediated by the remaining two Runx expansion of short-term HSCs (ST-HSCs) (Figures 2b and e). Further genes, which are also expressed in hematopoietic tissues and analysis of the myeloid progenitor compartment revealed a share common consensus DNA binding sites with Runx1. significant increase in granulocyte/macrophage progenitor and Supporting this argument, we recently demonstrated that Runx3 megakaryocyte/erythroid progenitor, though the former was not has a role in hematopoiesis as aged Runx3 cKO mice exhibited a 11 seen in Cbfbfl/fl;Mx1-Cre+ mice (Figure 2e). Due to the expansion, mild myeloproliferative disorder with HSPC expansion. The common myeloid progenitor compartment was relatively contribution by Runx2 remains unknown as Runx2 cKO mice have decreased in BM of Cbfb cKO mice (Figure 2c). Cbfb cKO mice not been examined for hematopoietic phenotypes. also showed a drastic depletion in the common lymphoid Comparison of the phenotypes of the Cbfb KO mice examined progenitor population (Figure 2d). in this study and those of conditional Cbfb-MYH11 KI mice appears helpful to dissect the leukemogenic mechanism of Cbfβ-SMMHC. Similar to the observation in BM, the spleen of Cbfb cKO mice 12 showed a dramatic increase in both KSL and myeloid progenitor Kuo et al. described differentiation blocks with an expansion of populations (Figure 2f), particularly in the ST-HSC population abnormal myeloid progenitor population (having an megakaryocyte/erythroid progenitor-like immunophenotype) in the condi- (Supplementary Figure 3A), concomitant with an increase in tional Cbfb-MYH11 KI mice. These phenotypes are largely similar to spleen weight (Figure 2g). These results suggest an increased those observed in our Cbfb cKO mice. Two most-upregulated extramedullary hematopoiesis in Cbfb cKO mice. genes in Cbfb-MYH11 KI cells, Csf2rb and Il1rl1,13 were also To investigate whether there was an increase in functional overexpressed in Cbfb cKO cells (Supplementary Figure 3D). In hematopoietic stem/progenitor cells (HSPCs), an in vitro colony contrast, HSC integrity was abrogated in Cbfb cKO mice, whereas it formation assay and an in vivo bone marrow transplantation was fully maintained in the conditional Cbfb-MYH11 KI mice. This were performed. A significant increase in colony forming activity fl/fl + result was further supported by the expression of Gata2 gene, was observed in the BM cells from Cbfb ;Mx1-Cre mice which is known to be critical for HSC maintenance:14 Gata2 was (Supplementary Figure 3B). The increased colony forming suppressed in the Cbfb cKO stem/progenitor cells (Supplementary activity of Cbfbfl/fl;Mx1-Cre+ HSPCs was maintained in subsequent fl/fl + Figure 3D), although reported to be overexpressed in the Cbfb- platings. Similar results were obtained with Cbfb ;Vav-iCre cells MYH11 KI cells.13 These results imply that the leukemogenic (data not shown). In striking contrast, results of the bone effects of Cbfβ-SMMHC may include fusion protein-specific, marrow transplantation assay showed that recipient mice stemness-related mechanisms other than simply inhibiting Cbfβ transplanted with Cbfb-deficient cells have extremely low function. donor chimerism in the various hematopoietic tissues, including Although both Cbfbfl/fl;Mx1-Cre+ and Cbfbfl/fl;Vav-iCre+ mice stem/progenitor fractions in the BM (Figure 2h; Supplementary exhibited two major phenotypes, some differences also exist. An Figure 3C and data not shown). Consistent with this observation, fl/fl fl fl obvious difference is the lethality observed in adult Cbfb ; aged Cbfb / ;Mx1-Cre+ mice show increased frequency of the Vav-iCre+ mice, but not in Cbfbfl/fl;Mx1-Cre+ mice (Figure 1c). The non-excised allele (Supplementary Figure 1C), suggesting that Vav promoter is activated at E10.5 onwards during the critical time Cbfb deficiency abrogates HSC integrity. These results suggest when the HSC population is rapidly expanding, while Mx1-Cre that while the progenitors in Cbfb cKO mice possess greater expression is induced at adult stage during the time when HSCs proliferative capacity, Cbfb-deficient HSCs were incapable of are largely maintained in quiescence (Supplementary Figure 4B). engraftment. Hence, Cbfb seems to be dispensable for hematopoiesis at the

Figure 1. Cbfb cKO mice show pancytopenia due to differentiation blocks. (a) Complete blood counts performed on Cbfbfl/fl;Mx1-Cre− (n = 17) and Cbfbfl/fl;Mx1-Cre+ (n = 25) at 4 weeks post induction, and Cbfbfl/fl;Vav-iCre− (n = 16) and Cbfbfl/fl;Vav-iCre+ mice (n = 19) at 4–6 weeks old. Mean ± s.d. of leukocyte, hemoglobin and platelet counts are shown. (b) Kaplan–Meier survival curves of Cbfbfl/fl;Mx1-Cre− (dotted line, n = 26), Cbfbfl/fl;Mx1-Cre+ (dashed line, n = 35), Cbfbfl/fl;Vav-iCre− (dotted line, n = 6) and Cbfbfl/fl;Vav-iCre+ (solid line, n = 6) mice. The P-value from Mantel- Cox test is shown. Vertical ticks represent censored cases. (c) Frequency of myeloid cells in BM of Cbfb cKO mice at either 4 weeks post induction or 4–6 weeks old. Mean ± s.d. are shown (Cbfbfl/fl;Mx1-Cre−, n = 5; Cbfbfl/fl;Mx1-Cre+, n = 5; Cbfbfl/fl;Vav-iCre−, n = 4; Cbfbfl/fl;Vav-iCre+, n = 5). Cbfb cKO mice exhibit a reduction of mature Mac1hiGr1hi granulocytes, accompanied by an expansion of Mac1hiGr1int myeloid cells in the BM. (d) May-Grünwald-Giemsa staining of BM cells in Cbfb cKO mice. Representative pictures of cells showing an increase in ring-shaped nuclei cells, corresponding to an immature granulocyte precursor population, in Cbfb cKO mice. (e) Frequency of B cells in BM of Cbfb cKO mice at either 4 weeks post induction or 4–6 weeks old. Mean ± SD are shown (Cbfbfl/fl;Mx1-Cre−, n = 5; Cbfbfl/fl;Mx1-Cre+, n = 5; Cbfbfl/fl;Vav-iCre−, n = 4; Cbfbfl/fl;Vav-iCre+, n = 4). Cbfb cKO mice exhibit a pronounced reduction of B220+CD19+ B cells in the BM due to a significant block in maturation from B220intIgD− to B220hiIgD+ B cells. No abnormalities were detected in the earlier developmental stages. (f) Weight of thymus in Cbfb cKO mice at either 4 weeks post induction or 4–6 weeks old. Mean ± s.d. of thymus weight are shown (Cbfbfl/fl;Mx1-Cre−, n = 7; Cbfbfl/fl; Mx1-Cre+, n = 7; Cbfbfl/fl;Vav-iCre−, n = 5; Cbfbfl/fl;Vav-iCre+, n = 6). (g, h) Frequency of T cells in thymus of Cbfb cKO mice at either 4 weeks post induction or 4 weeks old. Mean ± s.d. are shown (Cbfbfl/fl;Mx1-Cre−, n = 4; Cbfbfl/fl;Mx1-Cre+, n = 4; Cbfbfl/fl;Vav-iCre−, n = 2; Cbfbfl/fl;Vav-iCre+, n = 3). Cbfb cKO mice show a differentiation block from double-negative 1 (DN1) to DN2 population. In addition, Cbfbfl/fl;Vav-iCre+ mice show decreased DN4 populations. (i) Frequency of erythroid cells in BM of Cbfb cKO mice at either 4 weeks post induction or 4 weeks old. Mean ± s. d. are shown (Cbfbfl/fl;Mx1-Cre−, n = 5; Cbfbfl/fl;Mx1-Cre+, n = 5; Cbfbfl/fl;Vav-iCre−, n = 4; Cbfbfl/fl;Vav-iCre+, n = 5). Cbfb cKO mice show a significant increase in Ter119intCD71hi pro-erythroblast frequency with decreased Ter119hi erythroblasts. (j, k) Frequency of megakaryocyte cells in BM (j) and spleen (k)ofCbfb cKO mice at either 4 weeks post induction or 4 weeks old. Mean ± s.d. are shown (Cbfbfl/fl;Mx1-Cre−, n = 5; Cbfbfl/fl;Mx1- Cre+, n = 5; Cbfbfl/fl;Vav-iCre−, n = 4; Cbfbfl/fl;Vav-iCre+, n = 5). Cbfbfl/fl;Vav-iCre+ mice show an increase in CD41+CD61+ megakaryocytic lineage cells in both BM and spleen, while Cbfbfl/fl;Mx1-Cre+ mice showed an increase only in spleen. Asterisk(s) represents significant difference (*Po 0.05, **Po 0.01 and ***Po 0.001, Student’s t-test).

6 (Lin--gated) 60 * * Cbfbf/f Cbfbf/f;Mx1-Cre Cbfbf/f Cbfbf/f;Vav-iCre **

40.5 4.8 39.0 18.2 35.0 3.8 22.0 36.0 cells (%) 40 4 - *** *** * 20 2 c-Kit Frequency in BM (%)

Frequency in Lin 0 0 KSL Myeloid KSL Myeloid Sca-1 progenitors progenitors

Cbfbf/f (KSL-gated) f/f 100 * Cbfb ;Mx1-Cre Cbfbf/f Cbfbf/f;Mx1-Cre Cbfbf/f Cbfbf/f;Vav-iCre ** Cbfbf/f Cbfbf/f;Vav-iCre 24.6 6.9 37.3 31.8 80 60 ** ** 40 Flt3 9.5 53.8 0.4 71.7 * 9.3 66.7 17.3 76.0 20

Frequency in KSL cells (%) Frequency in KSL 0 CD34 LT-HSC ST-HSC MPP

(myeloid progenitor-gated) 80 f/f f/f f/f f/f Cbfb Cbfb ;Mx1-Cre Cbfb Cbfb ;Vav-iCre * *** 60 39.6 27.4 40.9 67.6 * *

R 40 *** 25.8 3.2 26.7 4.0 * Fc 20 31.5 60.7 31.8 26.8 progenitors (%)

Frequency in myeloid 0 CD34 CMP GMP MEP

*** 4 * 2.0 ** 0.20 - 30 * 3 * *** ** * Sca-1 2 * *** 1.5 * int 0.15 20 1 0.5 1.0

cells (%) 10 0.05 * 0.5 *** * *** Frequency in BM (%) Frequency in BM (%) Frequency in BM (%)

Frequency in c-Kit 0 0 0.0 0.00 CLP LT-HSC ST-HSC MPP CMPGMP MEP CLP

8 *** * BM PB 150 100 100 *** * *** *** 90 90 6 * *** 100 80 80 70 4 70 * 60 60 50 50 2 50

cells in PB (%) 1 * cells in BM (%) 1 Spleen weight (mg)

Frequency in spleen (%) 0 0 0 0 Frequency of CD45.2/CD45.2 f/f f/f f/f f/f f/f f/f Frequency of CD45.2/CD45.2 KSL Myeloid Cbfb Cbfb ; Cbfb Cbfb ; Cbfb Cbfb ; Cbfbf/f Cbfbf/f; progenitors Mx1-Cre Vav-iCre Vav-iCre Vav-iCre

adult stage, but is indispensable for the expansion of HSCs after those in Cbfbfl/fl;Mx1-Cre+ mice. This age-dependent phenotypic definitive HSCs have been generated from hemogenic endothe- enhancement may underlie the clinical observation that human lium. More importantly, the HSPC expansion and differentiation RUNX leukemia such as inv(16), t(8;21) and t(12;21) is prevalent in blocks in Cbfbfl/fl;Vav-iCre+ mice were more pronounced than childhood and younger adults.15

Figure 2. Cbfb cKO mice exhibit HSPC expansion. (a–e) Flow-cytometric analysis of the HSPC compartments in BM of Cbfb cKO mice at either 4 weeks post induction or 4–6 weeks old. Left, representative FACS plots of 0.2–1×106 cells gated on viable Lineage− cells (a), KSL cells (b) and c-Kit+Sca-1−Lineage− (myeloid progenitors) (c) are shown. Right, mean ± s.d. of percentage of KSL and myeloid progenitors (a), LT-HSC, ST-HSC and MPP compartments (b), CMP, GMP and MEP compartments (c) and CLP compartment (d) within their respective parent populations are shown (Cbfbfl/fl;Mx1-Cre−, n = 4; Cbfbfl/fl;Mx1-Cre+, n = 4; Cbfbfl/fl;Vav-iCre−, n = 4; Cbfbfl/fl;Vav-iCre+, n = 4). Mean ± s.d. of percentages of these populations in whole BM are also shown (a, far right and e). Three independent experiments were performed. LT-HSC, long-term hematopoietic stem cell (c-Kit+Sca-1+Lineage−CD34−Flt3−); ST-HSC, short-term hematopoietic stem cell (c-Kit+Sca-1+Lineage−CD34+Flt3−); MPP, multipotent progenitor (c-Kit+Sca-1+Lineage−CD34+Flt3+); CMP, common myeloid progenitor (c-Kit+Sca-1−Lineage−CD34+FcγRlo); GMP, granulocyte/macrophage progenitor (c-Kit+Sca-1−Lineage−CD34+FcγRhi); MEP, megakaryocyte/erythroid progenitor (c-Kit+Sca-1−Linea- ge−CD34−FcγRlo); CLP, common lymphoid progenitor (c-KitloSca-1loLineage−IL-7Rα+). (f) Flow-cytometric analysis of the HSPC compartment in spleen of Cbfb cKO mice at either 4 weeks post induction or 4–6 weeks old. Mean ± s.d. of percentage of KSL cells and myeloid progenitors in spleen is shown (Cbfbfl/fl;Mx1-Cre−, n = 4; Cbfbfl/fl;Mx1-Cre+, n = 4; Cbfbfl/fl;Vav-iCre−, n = 4; Cbfbfl/fl;Vav-iCre+, n = 4). (g) Weight of spleen in Cbfb cKO mice at either 4 weeks post induction or 4–6 weeks old. Mean ± s.d. of spleen weight are shown (Cbfbfl/fl;Mx1-Cre−, n = 7; Cbfbfl/fl;Mx1-Cre+, n = 7; Cbfbfl/fl;Vav-iCre−, n = 5; Cbfbfl/fl;Vav-iCre+, n = 6). (h) Frequency of CD45.2/CD45.2 donor cells in BM (left) and PB (right) at 8 weeks post transplantation. Mean ± s.d. are shown (Cbfbfl/fl;Vav-iCre−-transplanted, n = 3; Cbfbfl/fl;Vav-iCre+-transplanted, n = 4 (left), Cbfbfl/fl;Vav-iCre-- transplanted, n = 4; Cbfbfl/fl;Vav-iCre+-transplanted, n = 6 (right)). For the bone marrow transplantation (BMT) experiment, 8000 sorted donor BM KSL cells from Cbfbfl/fl;Vav-iCre− and Cbfbfl/fl;Vav-iCre+ mice (CD45.2/CD45.2) were transplanted, with 0.2x106 radioprotective BM cells (CD45.1/CD45.2), into sublethally irradiated C57BL/6 recipient mice (CD45.1/CD45.1). Asterisk(s) represent significant differences (*Po 0.05, **Po 0.01, ***Po 0.001, Student’s t-test).

CONFLICT OF INTEREST 2 Jacob B, Osato M, Yamashita N, Wang CQ, Taniuchi I, Littman DR et al. Stem cell The authors declare no conflict of interest. exhaustion due to Runx1 deficiency is prevented by Evi5 activation in leukemo- genesis. Blood 2010; 115: 1610–1620. 3 Growney JD, Shigematsu H, Li Z, Lee BH, Adelsperger J, Rowan R et al. Loss of ACKNOWLEDGEMENTS Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype. Blood 2005; 106:494–504. We thank M Mok, C Chung and HF Tan for their technical assistance; K Rajewshky for 4 Taniuchi I, Osato M, Egawa T, Sunshine MJ, Bae SC, Komori T et al. Differential Mx-Cre Tg mice; and members of MD2 Vivarium, NUS, for mouse husbandry. This requirements for Runx proteins in CD4 repression and epigenetic silencing during work was supported by A*STAR (Agency of Science, Technology and Research), T lymphocyte development. Cell 2002; 111: 621–633. Biomedical Research Council, National Medical Research Council, National Research 5 Osato M. Point mutations in the RUNX1/AML1 gene: another actor in RUNX Foundation Singapore and the Singapore Ministry of Education under its Research leukemia. Oncogene 2004; 23: 4284–4296. Centres of Excellence initiative. 6 Speck NA, Gilliland DG. Core-binding factors in haematopoiesis and leukaemia. Nat Rev Cancer 2002; 2:502–513. 7 Ley TJ, Miller C, Ding L, Raphael BJ, Mungall AJ, Robertson AG et al. Genomic and AUTHOR CONTRIBUTIONS epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med 2013; 368: 2059–2074. CQW and DWC performed experiments, analyzed data and wrote the manuscript; 8 Liu P, Tarle SA, Hajra A, Claxton DF, Marlton P, Freedman M et al. Fusion between JYC performed experiments and analyzed data; CWJ and IT provided research tools; transcription factor CBF beta/PEBP2 beta and a myosin heavy chain in acute VT and MO designed research and wrote the manuscript; and all authors reviewed myeloid leukemia. Science 1993; 261: 1041–1044. and approved the manuscript. 9 Castilla LH, Wijmenga C, Wang Q, Stacy T, Speck NA, Eckhaus M et al. Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos hetero- CQ Wang1,2,7, DWL Chin1,7, JY Chooi1,7, WJ Chng1, I Taniuchi3, zygous for a knocked-in leukemia gene CBFB-MYH11. Cell 1996; 87: 687–696. V Tergaonkar2 and M Osato1,4,5,6 10 Chen MJ, Yokomizo T, Zeigler BM, Dzierzak E, Speck NA. Runx1 is required for the 1Cancer Science Institute of Singapore, National University of endothelial to haematopoietic cell transition but not thereafter. Nature 2009; 457: – Singapore, Singapore; 887 891. 2 11 Wang CQ, Motoda L, Satake M, Ito Y, Taniuchi I, Tergaonkar V et al. Runx3 Institute of Molecular and Cell Biology, Singapore; fi 122 3 de ciency results in myeloproliferative disorder in aged mice. Blood 2013; : RIKEN Center for Integrative Medical Sciences, Yokohama, Japan; 562–566. 4 Institute of Bioengineering and Nanotechnology, Singapore; 12 Kuo YH, Landrette SF, Heilman SA, Perrat PN, Garrett L, Liu PP et al. 5 Department of Paediatrics, National University of Singapore, Cbf beta-SMMHC induces distinct abnormal myeloid progenitors able to develop Singapore and acute myeloid leukemia. Cancer Cell 2006; 9:57–68. 6International Research Center for Medical Sciences, Kumamoto 13HydeRK,KamikuboY,AndersonS,KirbyM,AlemuL,ZhaoLet al. Cbfb/Runx1 University, Kumamoto, Japan repression-independent blockage of differentiation and accumulation of E-mail: [email protected] or [email protected] Csf2rb-expressing cells by Cbfb-MYH11. Blood 2010; 115: 1433–1443. 7These authors contributed equally to this work. 14 de Pater E, Kaimakis P, Vink CS, Yokomizo T, Yamada-Inagawa T, van der Linden R et al. Gata2 is required for HSC generation and survival. J Exp Med 2013; 210: 2843–2850. REFERENCES 15 Mrozek K, Marcucci G, Nicolet D, Maharry KS, Becker H, Whitman SP et al. Prognostic significance of the European LeukemiaNet standardized system for 1 Wang Q, Stacy T, Miller JD, Lewis AF, Gu TL, Huang X et al. The CBFbeta subunit is reporting cytogenetic and molecular alterations in adults with acute myeloid essential for CBFalpha2 (AML1) function in vivo. Cell 1996; 87:697–708. leukemia. J Clin Oncol 2012; 30: 4515–4523.

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