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. 2014 May 12;25(5):652-65.
doi: 10.1016/j.ccr.2014.03.016. Epub 2014 May 1.

MLL3 Is a Haploinsufficient 7q Tumor Suppressor in Acute Myeloid Leukemia

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Free PMC article

MLL3 Is a Haploinsufficient 7q Tumor Suppressor in Acute Myeloid Leukemia

Chong Chen et al. Cancer Cell. .
Free PMC article

Abstract

Recurring deletions of chromosome 7 and 7q [-7/del(7q)] occur in myelodysplastic syndromes and acute myeloid leukemia (AML) and are associated with poor prognosis. However, the identity of functionally relevant tumor suppressors on 7q remains unclear. Using RNAi and CRISPR/Cas9 approaches, we show that an ∼50% reduction in gene dosage of the mixed lineage leukemia 3 (MLL3) gene, located on 7q36.1, cooperates with other events occurring in -7/del(7q) AMLs to promote leukemogenesis. Mll3 suppression impairs the differentiation of HSPC. Interestingly, Mll3-suppressed leukemias, like human -7/del(7q) AMLs, are refractory to conventional chemotherapy but sensitive to the BET inhibitor JQ1. Thus, our mouse model functionally validates MLL3 as a haploinsufficient 7q tumor suppressor and suggests a therapeutic option for this aggressive disease.

Figures

Figure 1
Figure 1. Chromosome 7 Loss or Deletion Is Associated with Mutations in NF1/RAS and TP53 Pathways
(A) ROMA plots depicting copy number changes of three AML cases. Data plotted are the normalized fluorescence log ratio for each probe (85 K). Top plot, whole genome view; left to right, chromosomes 1–22, X, Y. Bottom plots: high resolution of chromosome 17. (B) Copy number events of the AML samples with chromosome 7 or 7q deletions (−7/del(7q)) in TCGA AML cohort. Blue, deletion; red, amplification. (C) Top, heatmap of mutations in NF1/RAS pathways and TP53 in −7/del(7q) and MLL3 truncated (*) AML in the TCGA cohort. Black, deletions; red, gain-of-function mutations; blue, truncation or loss-of-function mutations. Bottom, the ratio of NF1/RAS and TP53 mutations in −7/del(7q) compared to AML samples without −7/del(7q). p value was calculated by chi-square test. See also Figure S1.
Figure 2
Figure 2. RNAi-Mediated Cosuppression of Mll3 and Nf1 Cooperates with p53 Loss to Promote Myeloid Leukemogenesis
(A) Schematic experimental design. p53−/− HSPC were co-infected with GFP-linked and mCherry-linked shRNAs and then transplanted into sublethally irradiated recipient mice. (B–D) The recipient mice were then monitored for disease in a variety of ways including overall survival (B) as well as WBC (C) and red blood cell (D) counts (n = 10, 8 weeks posttransplant or upon death of leukemia-bearing shMll3;shNf1;p53−/− recipients if they died before 8 weeks), showing mean ± SD. (E) After sacrifice, the BM was harvested and analyzed by flow cytometry to determine the frequency double negative, GFP+, mCherry+, and double positive cells, as compared to their frequency pre-injection (Pre). In all experiments, n = 10 except in (E) where n = 3–4. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S2.
Figure 3
Figure 3. Characterization of MNP AML
(A) Histological analysis of blood (a), liver (b), spleen (c), and BM (d) of an MNP recipient mouse. Scale bar: 12 μm for (a–c) and 30 μm for (d). (B) Representative flow cytometry profiles of GFP/mCherry double-positive cells from BM of MNP recipient mice versus control mice (shRen;shNf1;p53−/− are shown; all other control groups are similar, not shown). Left: lymphocyte markers B220 and CD3; right: myeloid marker Mac-1 and stem cell marker c-kit. (C) Survival of secondary transplant recipient mice of three independent MNP AML. n = 5 per group. (D) Histological analysis of secondary transplant recipient mice of MNP AML. (a) blood smears, (b) section of bone marrow, (c) section of spleen, and (d) section of liver. Scale bar: 12 μm for (a–c) and 30 μm for (d). See also Figure S3.
Figure 4
Figure 4. In Vivo CRISPR/Cas9 Confirmed that Mll3 Is a Haploinsufficient Tumor Suppressor in AML
(A) Schematic experimental design. p53−/− HSPC were transduced with mCherry-shNf1 and then CRISPR/Cas9 constructs targeting a control, noncoding region on chromosome 8 (cr_Ctrl) or Mll3 (cr_Mll3) were transiently introduced by electroporation, and transplanted into sublethally irradiated recipient mice. (B) The average survival and WBC counts of recipient mice transplanted with cr_Ctrl;shNf1;p53−/− and cr_Mll3;shNf1;p53−/− HSPC, showing mean ± SD (n = 3). (C) Blood smear and BM sections of recipient mice transplanted with cr_Ctrl;shNf1;p53−/− and cr_Mll3;shNf1;p53−/− HSPC. Scale bar: 12 μm. (D) Flow cytometry analysis of cr_Mll3;shNf1;p53−/− AMLs shows the expressions of mCherry and myeloid surface markers c-kit and Mac-1/Gr-1. (E) The sequences of the wild-type Mll3 region targeted by CRISPR/Cas9, and the resulting insertions/deletions detected in various cr_Mll3 leukemia clones. See also Figure S4.
Figure 5
Figure 5. Mll3 Inhibition Blocks HSPC Differentiation and Results in an MDS-like Syndrome in Mice
(A) Schematic experimental design. p53−/− HSPC were transduced with GFP– Ren or mCherry-linked Ren or Mll3 shRNAs. One day after infection, GFP+ and mCherry+ HSPC were mixed at a 1:1 ratio and transplanted into lethally irradiated syngeneic recipient mice. (B) The percentage of LT-HSC (Flt3linSca-1+c-kit+CD150+CD48CD34), ST-HSC (Flt3linSca-1+c-kit+CD150+CD48+CD34) and MPP (Flt3linSca-1+ c-kit+CD150-CD48+CD34) in the mCherry+ HSC (Flt3linSca1+c-kit+CD34) population at 6 weeks after transplant. n = 3. (C) The absolute numbers of LT-HSC, ST-HSC, and MPP in the BM of recipient mice at 6 weeks after transplant. n = 3. (D) The absolute numbers of common myeloid progenitor (CMP; Flt3linSca1c-kit+CD34+CD16/32), granulocyte-macrophage progenitor (GMP; Flt3lin Sca1c-kit+CD34+CD16/32+) and megakaryocyte erythrocyte progenitor (MEP; Flt3linSca1c-kit+CD34CD16/32) in the BM of recipient mice at 6 weeks after transplant. n = 3. (E) Left, the BM cellularity of shRen;p53−/− and shMll3;p53−/− mice at 6 weeks after transplant. Right, reconstitution ratio of mCherry+ donor cells in BM at 6 weeks after transplantation. n = 5. (F) White blood cell (WBC), red blood cell (RBC), and platelet (PLT) counts in shMll3 recipient mice compared to shRen control mice at 6 weeks after transplant. n = 5. (G) Reconstitution ratio of mCherry+ donor cells in the peripheral blood at 6 weeks after transplantation. n = 5. (H) Representative pictures showing dysplastic blood and BM cells from shMll3;p53−/− mice at 6 weeks after transplant. Howell Jolly body in peripheral blood (a); nucleated RBC in peripheral blood (b); hypersegmented neutrophil in peripheral blood (c); blast in peripheral blood (d); dysplastic megakaryocytes in BM (e). Scale bar: 5 μm. (I) Number of colonies formed per 10,000 BM cells from shRen;p53−/− or shMll3;p53−/− recipient mice 6 weeks after transplant. n = 3. (B–G) and (I) show mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S5.
Figure 6
Figure 6. Mll3 Suppression Enforces a Self-Renewal Gene Expression Program by Altering Chromatin Modifications
(A) Differentially expressed genes in shMll3;p53−/− HSPC compared to shRen;p53−/− HSPC (>1.5-fold different expression values, log2; p < 0.05 by two-way Student’s t test), as revealed by Illumina microarray gene expression analysis. (B) The correlation of the gene signatures between shMll3-1 and shMll3-2. The fold changes of the differently expressed genes (p < 0.05) in shMll3-1;p53−/− HSPC (versus shRen;p53−/− HSPC) and shMll3-1;p53−/− HSPC (versus shRen;p53−/− HSPC) were plotted as X and Y, respectively. k = 0.88 and Pearson’s coefficient R = 0.93. (C) qPCR confirmation of gene expression changes using cDNA of FACS purified LT-HSC (Flt3linSca-1+c-kit+CD150+CD48CD34) from shRen;p53−/− or shMll3;p53−/− recipient mice 6 weeks after transplant. n = 3. (D) Summary of the top functional categories of genes significantly enriched in shMll3;p53−/− HSPC. Analyses were performed on downregulated genes in shMll3;p53−/− HSPC, using DAVID (http://david.abcc.ncifcrf.gov/tools.jsp). (E) GSEA of shMll3;p53−/− HSPC expressing profile using a hematopoietic early progenitor-associated signature (NES = 1.89; FDR q = 0.0) and a mature hematopoietic cell-associated signature (NES = –2.22; FDR q = 0.0). (F) GSEA of shMll3;p53−/− HSPC expressing profile using a leukemic stem cell (LSC)-associated upregulated signature (NES = 1.48; FDR q = 0.02) and an LSC-associated downregulated signature (NES = −2.20; FDR q = 0.0). (G) Left, GSEA of shMll3;p53−/− HSPC expressing profile using a downregulated gene signature in human MDS HSC (versus normal HSC; NES = −2.17, FDR q = 0.0); right, GSEA of shMll3;p53−/− HSPC expressing profile using a downregulated gene signature in human −7/del(7q) MDS HSC (versus normal karyotype MDS HSC; NES = –2.14, FDR q = 0.0). (H) Upper, ChIP-qPCR showing the levels of H3K4me3 and K3K27me3 at loci of Gadd45 g, Il1r2 and Cpa3 in shRen;p53−/− and shMll3;p53−/− HSPC, with two biological repeats and two technical repeats for each sample. Lower, the locations of qPCR amplicons in target genes. (C) and (H) show mean ± SD. **p < 0.01; ***p < 0.001. See also Table S1.
Figure 7
Figure 7. Murine AMLs with Mll3 Suppression Are Resistant to Conventional Chemotherapy
(A and B) In vivo treatment of mice transplanted with shMll3;shNf1;p53−/− (MNP), MLL-AF9; NrasG12D (MAR) or AML1-ETO;NrasG12D (AER) AML with chemotherapy. Recipient mice were transplanted with MNP (n = 12), MAR (n = 12), or AER (n = 6 for veh, 7 for chemo), leukemic cells (CD45.2+) at day 0. Mice were treated with 100 mg/kg cytarabine (AraC) for 5 days and 3 mg/kg doxorubicin (Doxo) for 3 days by intraperitoneal injections starting at day 3 (MNP and MAR) or day 25 (AER) post-transplant. Kaplan-Meier survival curves of mice bearing MNP leukemias with or without chemotherapy treatment (A). Percentages of tumor cells in the bone marrow of terminal recipient mice, or at sacrifice 65 days after transplant in the case of the AER vehicle-treated group (B). n = 3. **p < 0.01, two-tail student t test. (C and D) AER AML cells were transduced with shRen, shp53, or shMll3 and then treated with indicated concentrations of AraC (C) or Doxo (D) for 3 days. Cell number was normalized to vehicle-treated cells. Graphs represent the average of four independent experiments and insets display half-maximal inhibitory concentration values. ***p < 0.001, two-way ANOVA test. (E) Dose response of MNP and MAR AML and MEFs to JQ1 in vitro. Cells were treated with vehicle or 1–200 nM JQ1 for 3 days and viable cells were counted by flow cytometry and cell numbers were normalized to vehicle-treated controls. Graph shows an average of three independent experiments. (F and G) MNP recipient mice were treated with vehicle or 50 mg/kg/day JQ1 by gavage for 1 week starting at 5 days after transplant. (F) left, WBC counts of MNP mice treated with vehicle or JQ1, 12 days after transplantation (n = 5). Right, blood smear of mice at day 12 after transplantation. Scale bar: 10 μm. (G) Survival curve of recipient mice. (H) Dose response of human AML cell lines to JQ1. Cells were treated with vehicle or 1–200 nM JQ1 for 3 days. Graphs represent the average of three independent experiments performed as described in (E). (C–F) and (H) show mean ± SD. See also Figure S6.

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