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

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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 © 2015 Macmillan Publishers Limited Leukemia (2015) 739 – 757 Letters to the Editor 754 * ** 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).
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