MicroRNA biogenesis is required for Myc-induced B-cell lymphoma development and survival

Abstract

Many tumor cells express globally reduced levels of microRNAs (miRNA), suggesting that decreased miRNA expression in premalignant cells contributes to their tumorigenic phenotype. In support of this, Dicer, an RNase III-like enzyme that controls the maturation of miRNA, was recently shown to function as a haploinsufficient tumor suppressor in nonhematopoietic cells. Because the Myc oncoprotein, a critical inducer of B-cell lymphomas, was reported to suppress the expression of multiple miRNAs in lymphoma cells, it was presumed that a deficiency of Dicer and subsequent loss of miRNA maturation would accelerate Myc-induced lymphoma development. We report here that, surprisingly, a haploinsufficiency of Dicer in B cells failed to promote B-cell malignancy or accelerate Myc-induced B-cell lymphomagenesis in mice. Moreover, deletion of Dicer in B cells of CD19-cre(+)/Emicro-myc mice significantly inhibited lymphomagenesis, and all lymphomas that did arise in these mice lacked functional Cre expression and retained at least one functional Dicer allele. Uncharacteristically, the lymphomas that frequently developed in the CD19-cre(+)/Dicer(fl/fl)/Emicro-myc mice were of very early precursor B-cell origin, a stage of B-cell development prior to Cre expression. Therefore, loss of Dicer function was not advantageous for lymphomagenesis, but rather, Dicer ablation was strongly selected against during Myc-induced B-cell lymphoma development. Moreover, deletion of Dicer in established B-cell lymphomas resulted in apoptosis, revealing that Dicer is required for B-cell lymphoma survival. Thus, Dicer does not function as a haploinsufficient tumor suppressor in B cells and is required for B-cell lymphoma development and survival.

Figure 1. CD19-cre⁺/Dicer^fl/fl/Eμ-myc mice have a protracted rate of lymphomagenesis, decreased precursor B cell numbers, and develop an altered type of lymphoma

Kaplan-Meier survival curves. (A) CD19-cre⁺/Dicer^fl/fl/Eμ-myc transgenic and CD19-cre^-/Dicer^fl/fl/Eμ-myc transgenic littermates; p=0.0001 (log-rank test). (B) CD19-cre⁺/Eμ-myc transgenic littermates with none, one, or two floxed Dicer alleles; p=0.0001 (log-rank test). The number (n) of mice in each group is denoted. (C) Representative dot plots of splenic B cells from the indicated mice (n=6 or 7 littermate matched pairs of each genotype). B220-APC versus IgM-FITC gated on total lymphocytes. (D) Dot plots of lymphoma cells from a CD19-cre⁺/Dicer^fl/fl/Eμ-myc transgenic mouse expressing markers of early precursor B cells. All plots are gated on total lymphocytes. Quadrants were set with fluorochrome-linked isotype controls.

Figure 2. Increased frequency of p53 deletions in CD19-cre⁺/Dicer^fl/fl/Eμ-myc lymphomas

(A) Protein lysates of lymphomas from CD19-cre⁺/Dicer^fl/fl/Eμ-myc transgenic mice were subjected to Western blot analysis for p53, ARF, Mdm2, and β-actin. Protein lysates from p53/Mdm2-double null MEFs and a B cell lymphoma containing mutant p53 were used as controls. (B) Southern blots for p53 and ARF of lymphomas from CD19-cre⁺/Dicer^fl/fl/Eμ-myc transgenic mice. Asterisks denote lymphomas that have deleted p53 and ARF. Lymphomas that contain p53 or ARF (plus signs) or that have deleted p53 (p53 Del) or ARF (ARF Del) are denoted.

Figure 3. Decreased CD19 expression and frequent loss of Cre expression in CD19-cre⁺/Dicer^fl/fl/Eμ-myc lymphomas

(A) Dot plots of CD19, B220, and IgM expression in two CD19-cre⁺/Dicer^fl/fl/Eμ-myc lymphomas. Quadrants were set with isotype controls for each lymphoma. For histograms, CD19 expression is grey and the isotype control for each is indicted with a dotted line. (B & C) qRT-PCR for CD19 (B) and Cre (C) expression relative to β-actin expression in lymphomas from the indicated genotype. (D) Western blots for Cre and β-actin in protein lysates of CD19-cre⁺/Eμ-myc lymphomas with none, one, or two floxed Dicer alleles. Protein lysates from lymphomas from CD19-cre⁺/Dicer^+/+/Eμ-myc lymphomas (+) and CD19-cre^-/Dicer^fl/fl/Eμ-myc lymphomas (-) are indicated.

Figure 4. CD19-cre⁺/Dicer^fl/fl/Eμ-myc transgenic lymphomas retain at least one Dicer allele

(A) Schematic of the Dicer locus with loxP sites denoted with open arrowheads. Location of the sites of primer pairs used to detect a region of Dicer by PCR are indicated with arrows. Size in base pairs (bp) of the PCR products from the specific primers is indicated. (A & B) Detection of conditional deleted and floxed Dicer alleles by PCR from genomic DNA from frozen CD19-cre⁺/Dicer^+/fl/Eμ-myc lymphomas (A), frozen CD19-cre⁺/Dicer^fl/fl/Eμ-myc lymphomas (B, left panel), or cultured CD19-cre⁺/Dicer^fl/fl/Eμ-myc lymphomas (B, right panel). Genomic DNA from the tail of a Dicer^+/fl mouse with a floxed Dicer allele is a control (A). Genomic DNA from Dicer^fl/fl (D^fl/fl) mouse embryo fibroblasts (MEF) with or without Cre recombinase serves as controls for Dicer conditional deletion (B). (C) qRT-PCR for Dicer in individual Eμ-myc lymphomas of the indicated genotype. A Dicer^fl/fl (D^fl/fl) MEF expressing Cre recombinase serves as a control for Dicer conditional deletion. (C) Taqman qRT-PCR for miR-20a or miR-106a in the indicated Eμ-myc lymphomas (other miRNA also evaluated with similar results). A Dicer^fl/fl (D^fl/fl) MEF expressing Cre recombinase serves as a control for loss of miRNA expression.

Figure 5. Dicer loss induces apoptosis of established B cell lymphomas

(A-E) Lymphomas from Dicer^fl/fl/Eμ-myc transgenic mice infected with CreER^T2 encoding retrovirus or an empty retrovirus (Vec). 4-OHT or vehicle control (ETOH) was added to lymphoma cell cultures at day or time 0 and cell number (A), viability (B), apoptosis (percentage of cells in subG1 indicated, C), and Dicer gene rearrangement (D) were evaluated at intervals. A, B, and C contain representative data from a minimum of three independent experiments. For D, data for four independent Dicer^fl/fl/Eμ-myc CreER^T2 expressing lymphomas are shown. (E) Dicer rearrangement PCR of FACS sorted single lymphoma clones that emerged following 4-OHT activation of CreER^T2. Nineteen of 43 total clones analyzed are shown. For D and E, a Dicer^fl/fl (D^fl/fl) MEF with or without Cre recombinase serves as a control for Dicer deletion.