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MYBL2 is a sub-haploinsufficient tumor suppressor gene in myeloid malignancy.

Heinrichs S, Conover LF, Bueso-Ramos CE, Kilpivaara O, Stevenson K, Neuberg D, Loh ML, Wu WS, Rodig SJ, Garcia-Manero G, Kantarjian HM, Look AT - Elife (2013)

Bottom Line: Here we show that MYBL2, a gene within the 20q CDR, is expressed at sharply reduced levels in CD34+ cells from most MDS cases (65%; n = 26), whether or not they harbor 20q abnormalities.In a murine competitive reconstitution model, Mybl2 knockdown by RNAi to 20-30% of normal levels in multipotent hematopoietic progenitors resulted in clonal dominance of these 'sub-haploinsufficient' cells, which was reflected in all blood cell lineages.We conclude that downregulation of MYBL2 activity below levels predicted by classical haploinsufficiency underlies the clonal expansion of hematopoietic progenitors in a large fraction of human myeloid malignancies.

View Article: PubMed Central - PubMed

Affiliation: Institute of Transfusion Medicine , University Hospital Essen , Essen , Germany ; Department of Pediatric Oncology , Dana-Farber Cancer Institute , Boston , United States.

ABSTRACT
A common deleted region (CDR) in both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) affects the long arm of chromosome 20 and has been predicted to harbor a tumor suppressor gene. Here we show that MYBL2, a gene within the 20q CDR, is expressed at sharply reduced levels in CD34+ cells from most MDS cases (65%; n = 26), whether or not they harbor 20q abnormalities. In a murine competitive reconstitution model, Mybl2 knockdown by RNAi to 20-30% of normal levels in multipotent hematopoietic progenitors resulted in clonal dominance of these 'sub-haploinsufficient' cells, which was reflected in all blood cell lineages. By 6 months post-transplantation, the reconstituted mice had developed a clonal myeloproliferative/myelodysplastic disorder originating from the cells with aberrantly reduced Mybl2 expression. We conclude that downregulation of MYBL2 activity below levels predicted by classical haploinsufficiency underlies the clonal expansion of hematopoietic progenitors in a large fraction of human myeloid malignancies. DOI:http://dx.doi.org/10.7554/eLife.00825.001.

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Mybl2 knockdown in 32D cells by a series of shRNAs.Mybl2 knockdown in 32D cells by a series of shRNAs (control shRNAs are labeled B and C; Mybl2-specific shRNAs are labeled M1–M5). Mybl2 expression levels were measured by qRT-PCR normalized to three control genes (upper panel) and by Western blotting with tubulin as a normalization control (lower panel).DOI:http://dx.doi.org/10.7554/eLife.00825.015
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fig5s1: Mybl2 knockdown in 32D cells by a series of shRNAs.Mybl2 knockdown in 32D cells by a series of shRNAs (control shRNAs are labeled B and C; Mybl2-specific shRNAs are labeled M1–M5). Mybl2 expression levels were measured by qRT-PCR normalized to three control genes (upper panel) and by Western blotting with tubulin as a normalization control (lower panel).DOI:http://dx.doi.org/10.7554/eLife.00825.015

Mentions: If sub-haploinsufficient decreases in MYBL2 dosage contribute to MDS pathogenesis, a technique such as RNAi knockdown with a series of shRNA hairpins of graded potencies should provide an experimental model that can be used to demonstrate a clonal advantage within hematopoietic progenitor cells and implicate the level of Mybl2 knockdown that produces the maximum effect. Thus, using five independent, specific shRNA-expressing vectors to target Mybl2 in primitive hematopoietic cells, we established a competitive reconstitution assay in mice. These vectors downregulated Mybl2 expression to levels that ranged from 13% (M1) to 33% (M5) of those expressed by control shRNA-expressing vectors in 32D cells, a murine myeloid cell line (Figure 5—figure supplement 1). After transducing Lin− mononuclear bone marrow cells in separate vials with these five vectors, which coexpressed a green fluorescent protein (GFP), or with two different control shRNA vectors coexpressing a red fluorescent protein (RFP) (Strack et al., 2008), we washed the cells, pooled the five Mybl2-specific shRNA knockdown cell aliquots and the two control aliquots, and transplanted equal numbers of these two cell pools into lethally irradiated mice (Figure 5A). A second group of mice were transplanted with pooled GFP+ and RFP+ cells expressing control shRNAs only, generating control/GFP vs control/RFP recipients (Figure 5—figure supplement 2A). Analysis of GFP and RFP levels in peripheral blood at 12 weeks (Figure 5B) showed a strikingly higher median frequency of GFP+ vs RFP+ cells in the peripheral blood of the mice reconstituted with GFP+/Mybl2-shRNA vs RFP+/control shRNA (30.9% vs 2.6%, p<0.0001). By contrast, in mice injected with control/GFP vs control/RFP cells, the median frequencies of both RFP- and GFP-expressing cells were low (1.5% and 3.4%; Figure 5—figure supplement 2B).10.7554/eLife.00825.014Figure 5.Competitive in vivo reconstitution assay testing expansion capacity of cells with low Mybl2 expression.


MYBL2 is a sub-haploinsufficient tumor suppressor gene in myeloid malignancy.

Heinrichs S, Conover LF, Bueso-Ramos CE, Kilpivaara O, Stevenson K, Neuberg D, Loh ML, Wu WS, Rodig SJ, Garcia-Manero G, Kantarjian HM, Look AT - Elife (2013)

Mybl2 knockdown in 32D cells by a series of shRNAs.Mybl2 knockdown in 32D cells by a series of shRNAs (control shRNAs are labeled B and C; Mybl2-specific shRNAs are labeled M1–M5). Mybl2 expression levels were measured by qRT-PCR normalized to three control genes (upper panel) and by Western blotting with tubulin as a normalization control (lower panel).DOI:http://dx.doi.org/10.7554/eLife.00825.015
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3713455&req=5

fig5s1: Mybl2 knockdown in 32D cells by a series of shRNAs.Mybl2 knockdown in 32D cells by a series of shRNAs (control shRNAs are labeled B and C; Mybl2-specific shRNAs are labeled M1–M5). Mybl2 expression levels were measured by qRT-PCR normalized to three control genes (upper panel) and by Western blotting with tubulin as a normalization control (lower panel).DOI:http://dx.doi.org/10.7554/eLife.00825.015
Mentions: If sub-haploinsufficient decreases in MYBL2 dosage contribute to MDS pathogenesis, a technique such as RNAi knockdown with a series of shRNA hairpins of graded potencies should provide an experimental model that can be used to demonstrate a clonal advantage within hematopoietic progenitor cells and implicate the level of Mybl2 knockdown that produces the maximum effect. Thus, using five independent, specific shRNA-expressing vectors to target Mybl2 in primitive hematopoietic cells, we established a competitive reconstitution assay in mice. These vectors downregulated Mybl2 expression to levels that ranged from 13% (M1) to 33% (M5) of those expressed by control shRNA-expressing vectors in 32D cells, a murine myeloid cell line (Figure 5—figure supplement 1). After transducing Lin− mononuclear bone marrow cells in separate vials with these five vectors, which coexpressed a green fluorescent protein (GFP), or with two different control shRNA vectors coexpressing a red fluorescent protein (RFP) (Strack et al., 2008), we washed the cells, pooled the five Mybl2-specific shRNA knockdown cell aliquots and the two control aliquots, and transplanted equal numbers of these two cell pools into lethally irradiated mice (Figure 5A). A second group of mice were transplanted with pooled GFP+ and RFP+ cells expressing control shRNAs only, generating control/GFP vs control/RFP recipients (Figure 5—figure supplement 2A). Analysis of GFP and RFP levels in peripheral blood at 12 weeks (Figure 5B) showed a strikingly higher median frequency of GFP+ vs RFP+ cells in the peripheral blood of the mice reconstituted with GFP+/Mybl2-shRNA vs RFP+/control shRNA (30.9% vs 2.6%, p<0.0001). By contrast, in mice injected with control/GFP vs control/RFP cells, the median frequencies of both RFP- and GFP-expressing cells were low (1.5% and 3.4%; Figure 5—figure supplement 2B).10.7554/eLife.00825.014Figure 5.Competitive in vivo reconstitution assay testing expansion capacity of cells with low Mybl2 expression.

Bottom Line: Here we show that MYBL2, a gene within the 20q CDR, is expressed at sharply reduced levels in CD34+ cells from most MDS cases (65%; n = 26), whether or not they harbor 20q abnormalities.In a murine competitive reconstitution model, Mybl2 knockdown by RNAi to 20-30% of normal levels in multipotent hematopoietic progenitors resulted in clonal dominance of these 'sub-haploinsufficient' cells, which was reflected in all blood cell lineages.We conclude that downregulation of MYBL2 activity below levels predicted by classical haploinsufficiency underlies the clonal expansion of hematopoietic progenitors in a large fraction of human myeloid malignancies.

View Article: PubMed Central - PubMed

Affiliation: Institute of Transfusion Medicine , University Hospital Essen , Essen , Germany ; Department of Pediatric Oncology , Dana-Farber Cancer Institute , Boston , United States.

ABSTRACT
A common deleted region (CDR) in both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) affects the long arm of chromosome 20 and has been predicted to harbor a tumor suppressor gene. Here we show that MYBL2, a gene within the 20q CDR, is expressed at sharply reduced levels in CD34+ cells from most MDS cases (65%; n = 26), whether or not they harbor 20q abnormalities. In a murine competitive reconstitution model, Mybl2 knockdown by RNAi to 20-30% of normal levels in multipotent hematopoietic progenitors resulted in clonal dominance of these 'sub-haploinsufficient' cells, which was reflected in all blood cell lineages. By 6 months post-transplantation, the reconstituted mice had developed a clonal myeloproliferative/myelodysplastic disorder originating from the cells with aberrantly reduced Mybl2 expression. We conclude that downregulation of MYBL2 activity below levels predicted by classical haploinsufficiency underlies the clonal expansion of hematopoietic progenitors in a large fraction of human myeloid malignancies. DOI:http://dx.doi.org/10.7554/eLife.00825.001.

Show MeSH
Related in: MedlinePlus