MLL-AF9-induced leukemogenesis requires coexpression of the wild-type Mll allele - PubMed (original) (raw)

MLL-AF9-induced leukemogenesis requires coexpression of the wild-type Mll allele

Austin T Thiel et al. Cancer Cell. 2010.

Abstract

Oncogenic fusion proteins are capable of initiating tumorigenesis, but the role of their wild-type counterparts in this process is poorly understood. The mixed lineage leukemia (MLL) gene undergoes chromosomal translocations, resulting in the formation of oncogenic MLL fusion proteins (MLL-FPs). Here, we show that menin recruits both wild-type MLL and oncogenic MLL-AF9 fusion protein to the loci of HOX genes to activate their transcription. Wild-type MLL not only catalyzes histone methylation at key target genes but also controls distinct MLL-AF9-induced histone methylation. Notably, the wild-type Mll allele is required for MLL-AF9-induced leukemogenesis and maintenance of MLL-AF9-transformed cells. These findings suggest an essential cooperation between an oncogene and its wild-type counterpart in MLL-AF9-induced leukemogenesis.

2010 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Menin is required for both H3K4 trimethylation and H3K79 dimethylation at Hoxa9 in MA9-transformed cells

(A) Western blot for menin in control or Men1 excised MA9-transformed AT1 cells, which harbored Men1f/f;Cre-ER. The cells were treated with either control DMSO (Men1f/f) or 4-OHT (_Men1_Δ/Δ) to excise the floxed Men1. (B–D) ChIP assay, with two distinct amplicons, for detecting dimethylated H3K79 (B), trimethylated H3K4 (C), and menin binding (D) at Hoxa9 in Men1f/f and _Men1_Δ/Δ AT1 cells. Error bars denote +/− SD.

Figure 2

Figure 2. Wt MLL and MA9 are recruited to Hoxa9 in a menin-dependent manner

AT1 cells were treated with either DMSO (Men1f/f) or 4-OHT (_Men1_Δ/Δ) and processed for ChIP assay with either (A) anti-MLL-C or (B) anti-AF9 C-terminus antibodies. (C) THP-1 cells were transduced with either control scrambled or Men1 shRNA-expressing lentiviruses, and used for ChIP assay with anti-AF9 or anti-Dot1L antibodies. Error bars denote +/− SD. See also Figure S1.

Figure 3

Figure 3. Wt Mll is required for growth of MA9-transformed leukemia cells and expression of Hoxa9 and CCNA2

(A) A diagram for the structure of wt MLL, MA9 fusion protein, and shRNAs targeting various parts of MLL-C but not MA9. AT1 cells were transduced with either vector or MLL-C shRNA 11 retroviruses and monitored for cell number (+/− SD) (B), wt Mll, Hoxa9, and cyclin A (CCNA2) mRNAs (C), and the protein levels of MLL-C and cyclin A (+/− SD) (D). THP-1 cells were transduced with either control scrambled shRNA lentiviruses (Scram) or MLL-C shRNAs. The resulting cells were monitored for change in number (+/− SD) (E), the mRNA levels of wt Mll, Hoxa9, and CCNA2 (+/− SD) (F), and the protein levels of MLL-C and cyclin A (G), MLL-N, MLL-AF9 (MA9), and menin (H). See also Figure S2.

Fig. 4

Fig. 4. Wt Mll excision reduces the number of MA9-transformed cells and Hox gene expression

(A) A diagram for the floxed Mll and the primers used to detect the intact or excised Mll. (B) 4-OHT induced excision of the floxed Mll. Spleen cells from a Mllf/f;Cre-ER mouse were cultured with either DMSO or 4-OHT, followed by isolation of the genomic DNA and PCR amplification. (C) A growth curve for MA9-transformed BM cells with either Mllf/f or _Mll_Δ/Δ (+/− SD, cells seeded in triplicate) (D) Quantification of Hoxa9 and Hoxa5 mRNAs in either Mllf/f or _Mll_Δ/Δ MA9-transformed cells (+/− SD). See also Figure S3.

Figure 5

Figure 5. Wt MLL is crucial for maximal methylation of both H3K4 and H3K79 at key target genes

THP-1 cells were transduced with either control scrambled shRNA or MLL-C shRNA 14 lentiviruses to knock down wt MLL and evaluated using ChIP assay for MLL-C binding to Hoxa9 (A) and for histone H3K4m3 and H3K79m2 at Hoxa9 (B) and CCNA2 (C). The specificity of the anti-H3K4m3 antibody and the anti-H3K79m2 were confirmed using specifically modified peptides and Western blot. Error bars denote +/− SD. See also Figure S4.

Figure 6

Figure 6. Wt MLL knockdown suppresses colony formation of MA9-transduced BM

(A) Procedure for the colony formation assay. Bone marrow (BM) cells from a C57B6 mouse were transduced with pMSCV-MA9 retroviruses, and replated in triplicate weekly in methylcellulose medium with G418. After the second plating, surviving MA9 cells were transduced with each of the MLL-C shRNAs (12 and 14) or scrambled vector. (B) A summary of colony numbers for control or Mll shRNA-transduced BM. (C) Representative colonies from the culture plates (Scale bars 5mM). Error bars denote +/− SD.

Figure 7

Figure 7. Wt Mll is required for colony formation of MA9-induced BM

(A) A flowchart for procedures of MA9-induced transformation and 4-OHT-induced Mll excision. (B) Mll excision reduced the number of colonies from MA9-transduced BM from the Mllf/f;Cre-ER mice. (C) 4-OHT failed to reduce colony formation of MA9 retrovirus-transduced BM from the Mllf/f mice. (D) Genotyping with genomic DNA showed that 4-OHT induced Mll excision in MA9-transformed cells with the Mllf/f;Cre-ER genotype (lane 2) but failed to induce Mll excision in MA9-transformed BM cells with Mllf/f but without the Cre-ER transgene (lane 4). (E) Wt Mll excision failed to reduce Hoxa9/Meis1-induced BM colony formation (Top). 4-OHT-induced wt Mll excision in Hoxa9/Meis1-transformed BM (Bottom). (F) Wt Mll excision reduced colony formation from normal BM. BM from corn oil (CO) or TAM-treated Mllf/f;Cre-ER mice was plated on methylcellulose medium and the colony number was scored at the first plating (Top). Wt Mll was excised in BM from TAM-treated, but not from corn oil-fed, Mllf/f;Cre-ER mice (Bottom). (G) BM from corn oil or TAM-treated Mllf/f;Cre-ER mice was first transduced with MA9, followed by plating on methylcellulose medium, and the colony number was scored at the first plating. Error bars denote +/− SD.

Figure 8

Figure 8. Wt Mll is required for MA9-induced leukemogenesis in mice

(A) A diagram for MA9-induced leukemogenesis and wt Mll excision in mice. TAM feeding was done 3 weeks after BM transplantation. (B) The total peripheral blood white blood cells (WBCs) in mice transplanted with MA9-transduced Mllf/f;Cre-ER BM were measured five weeks after corn oil or TAM feeding. (C) Flow cytometry analysis of peripheral blood CD11b+ MA9-transformed donor cells from transplanted mice, five weeks after the mice were fed with corn oil or TAM. (D) Flow cytometry analysis of CD45.2+ splenocytes from either terminally ill and corn oil-fed mice or TAM-fed mice to detect the percentage of c-kit high cells that were FcRγII/III+/CD34+. (E) A summary of the percentage of c-kit high cells that were FcRγII/III+/CD34+ from the corn oil or TAM-fed mice. (F) TAM-induced Mll excision in transplanted MA9-transduced BM in recipient mice increased the survival rate of the mice. Kaplan Meier curve for mice (C57B6/B6-SJL F1) transplanted with MA9-transduced Mllf/f;Cre-ER BM that were fed with either control corn oil (n=11) or TAM (n=7), 3 weeks after MA9 BM transplantation. (G) A model for menin, wt MLL, and MA9/Dot1L tripartite complex-controlled regulation of coupled yet distinct histone methylations, gene transcription, MA9-induced LSCs, and leukemogenesis. See also Figure S5.

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