TO THE EDITOR:

Multiple myeloma (MM) is a plasma cell neoplasm that remains incurable despite the introduction of novel agents. Therefore, it is necessary to decode the molecular mechanisms of myelomagenesis. Recent advances in next-generation sequencing technologies have highlighted the significance of known events that drive MM, and have also identified novel genetic alterations in MM.1 Importantly, DIS3 mutations have been identified in ∼10% of patients with MM, and loss of heterozygosity at chromosome 13q that leads to deletion of 1 allele of DIS3 has been observed in ∼40% of patients with MM.1,2 Furthermore, DIS3 mutations/deletions are associated with poor prognosis in MM.2 DIS3 is a member of the RNase II/R family. It is a catalytic subunit of RNA exosome that mediates degradation and processing of various RNAs.3 Recent studies have shown that Dis3 is required for immunoglobulin gene rearrangements, indicating its role in B cells.4,5 However, the roles of DIS3 in hematopoiesis and myelomagenesis remain incompletely understood.

To explore the functions of Dis3 in hematopoiesis, we first generated C57BL/6 mice carrying the floxed allele of Dis3 by introducing a pair of loxP sequences into introns 2 and 4 of the Dis3 gene in fertilized eggs using a clustered regularly interspaced short palindrome repeats (CRISPR)-Cas9 system (supplemental Figure 1A). We confirmed insertion of loxP sites by polymerase chain reaction genotyping and Sanger sequencing (supplemental Figure 1B-C). We then set out to generate hematopoietic cell–specific Dis3 knockout (KO) mice by crossing Dis3fl/fl mice and Mx-Cre mice. To avoid possible effects of Dis3 deficiency on nonhematopoietic cells, we transplanted bone marrow (BM) cells from Dis3fl/fl and Dis3fl/fl;Mx-Cre mice into lethally irradiated CD45.1+ recipient mice. Four weeks after transplantation, we deleted Dis3 by inducing Cre recombinase through an intraperitoneal injection of polyinosinic-polycytidylic acid (pIpC). We hereafter referred to the recipient mice recovered with the BM cells from Dis3fl/fl and Dis3fl/fl; Mx-Cre mice as wild type (WT) and Dis3Δ/ΔMx mice, respectively (Figure 1A). We confirmed generation of the Dis3 KO allele by genomic polymerase chain reaction (Figure 1B) and validated reduced Dis3 messenger RNA expression in the BM cells of Dis3Δ/ΔMx mice (Figure 1C). Of note, loss of Dis3 in hematopoietic cells resulted in severe pancytopenia at 2 weeks after Dis3 deletion (Figure 1D). Consistently, Dis3Δ/ΔMx mice exhibited markedly reduced BM cell counts with relative preservation of mature lymphocytes (Figure 1E-F). Detailed flow cytometric analysis of the BM cells revealed lower frequency of LineageSca-1+c-Kit+ (LSK) cells, hematopoietic stem cells (HSCs: CD150+CD48CD135 LSK), and multipotent progenitors (MPPs) in Dis3Δ/ΔMx mice than in WT mice (Figure 1G). The frequency of the 3 MPP populations (MPPMegakaryocyte (Mk)/Erythroid (E), MPPGranulocyte (G)/Monocyte (M), and MPPLymphoid (Ly)) that retain multilineage potential despite undergoing specific lineage priming6 was also significantly lower in Dis3Δ/ΔMx mice than in WT mice (Figure 1G). Dis3Δ/ΔMx mice also exhibited a significant decrease in the frequency of common myeloid progenitor (CMP), granulocyte/macrophage progenitor (GMP), and megakaryocyte/erythroid progenitor (MEP) cells, compared with that in WT mice (Figure 1H). In contrast, the frequency of common lymphoid progenitor (CLP) cells was comparable between WT and Dis3Δ/ΔMx mice (Figure 1H), whereas the absolute number of CLP cells was significantly reduced in Dis3Δ/ΔMx mice compared with in WT mice (Figure 1I). These results indicate that Dis3 is essential for normal hematopoiesis.

Figure 1.

Depletion of Dis3 severely compromises hematopoiesis. (A) Experimental schema for generating hematopoietic cell–specific Dis3 KO mice. Total BM cells (5 × 106 cells) isolated from Dis3fl/fl or Dis3fl/fl;Mx-Cre mice were transplanted into lethally irradiated CD45.1 recipient mice. Four weeks after transplantation, Cre recombinase was induced by intraperitoneal injections of 300 μg of pIpC on days 0, 2, and 4. The recipient mice recovered with Dis3fl/fl and Dis3fl/fl;Mx-Cre BM cells and, followed by Cre recombination, were referred to as WT and Dis3Δ/ΔMx, respectively. (B and C) On day 5, BM cells were collected from the recipient mice reconstituted with Dis3fl/fl (WT) and Dis3fl/fl;Mx-Cre (Dis3Δ/ΔMx) BM cells. (B) Generation of KO allele was assessed by genomic polymerase chain reaction (PCR; n = 3). (C) Dis3 messenger RNA expression in BM cells was quantified by quantitative PCR (n = 3-4). (D) Complete blood cell counts of WT and Dis3Δ/ΔMx mice (n = 9-10) 2 weeks after Dis3 depletion. (E) BM cell counts of WT and Dis3Δ/ΔMx mice (n = 9-10) 2 weeks after Dis3 depletion ( 2F2T: 2 femurs and 2 tibias). (F) Frequency of T cells (CD4+/CD8+), B cells (B220+), and myeloid cells (CD11b+) among CD45.2+ cells in the BM of WT and Dis3Δ/ΔMx mice (n = 5) 2 weeks after Dis3 depletion. (G) Frequency of LSK cells, HSCs (CD150+CD48CD135LSK), and MPPs (MPP: CD150CD48CD135LSK; MPPMk/E: CD150+CD48+CD135LSK; MPPG/M: CD150CD48+CD135LSK; and MPPLy: CD150CD48+CD135+LSK, as previously defined6) in the BM cells of WT and Dis3Δ/ΔMx mice (n = 9) 2 weeks after Dis3 depletion. (H) Frequency of CMP (LinSca-1c-Kit+CD34+FcγRlow), GMP (LinSca-1c-Kit+CD34+FcγRhigh), MEP (LinSca-1c-Kit+CD34FcγRlow), and CLP (IL-7Rα+LinSca-1lowc-Kitlow) cells among CD45.2+ cells in the BM of WT and Dis3Δ/ΔMx mice (n = 6) 2 weeks after Dis3 depletion. (I) Absolute number of CLP cells among CD45.2+ cells in the BM of WT and Dis3Δ/ΔMx mice (n = 6) 2 weeks after Dis3 depletion. (C-I) Data are mean ± standard error of the mean; ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001; 2-tailed t test. Animal studies were performed in accordance with a protocol approved by the Kumamoto University Institutional Animal Care and Use Committee. BMT, bone marrow transplantation.

Figure 1.

Depletion of Dis3 severely compromises hematopoiesis. (A) Experimental schema for generating hematopoietic cell–specific Dis3 KO mice. Total BM cells (5 × 106 cells) isolated from Dis3fl/fl or Dis3fl/fl;Mx-Cre mice were transplanted into lethally irradiated CD45.1 recipient mice. Four weeks after transplantation, Cre recombinase was induced by intraperitoneal injections of 300 μg of pIpC on days 0, 2, and 4. The recipient mice recovered with Dis3fl/fl and Dis3fl/fl;Mx-Cre BM cells and, followed by Cre recombination, were referred to as WT and Dis3Δ/ΔMx, respectively. (B and C) On day 5, BM cells were collected from the recipient mice reconstituted with Dis3fl/fl (WT) and Dis3fl/fl;Mx-Cre (Dis3Δ/ΔMx) BM cells. (B) Generation of KO allele was assessed by genomic polymerase chain reaction (PCR; n = 3). (C) Dis3 messenger RNA expression in BM cells was quantified by quantitative PCR (n = 3-4). (D) Complete blood cell counts of WT and Dis3Δ/ΔMx mice (n = 9-10) 2 weeks after Dis3 depletion. (E) BM cell counts of WT and Dis3Δ/ΔMx mice (n = 9-10) 2 weeks after Dis3 depletion ( 2F2T: 2 femurs and 2 tibias). (F) Frequency of T cells (CD4+/CD8+), B cells (B220+), and myeloid cells (CD11b+) among CD45.2+ cells in the BM of WT and Dis3Δ/ΔMx mice (n = 5) 2 weeks after Dis3 depletion. (G) Frequency of LSK cells, HSCs (CD150+CD48CD135LSK), and MPPs (MPP: CD150CD48CD135LSK; MPPMk/E: CD150+CD48+CD135LSK; MPPG/M: CD150CD48+CD135LSK; and MPPLy: CD150CD48+CD135+LSK, as previously defined6) in the BM cells of WT and Dis3Δ/ΔMx mice (n = 9) 2 weeks after Dis3 depletion. (H) Frequency of CMP (LinSca-1c-Kit+CD34+FcγRlow), GMP (LinSca-1c-Kit+CD34+FcγRhigh), MEP (LinSca-1c-Kit+CD34FcγRlow), and CLP (IL-7Rα+LinSca-1lowc-Kitlow) cells among CD45.2+ cells in the BM of WT and Dis3Δ/ΔMx mice (n = 6) 2 weeks after Dis3 depletion. (I) Absolute number of CLP cells among CD45.2+ cells in the BM of WT and Dis3Δ/ΔMx mice (n = 6) 2 weeks after Dis3 depletion. (C-I) Data are mean ± standard error of the mean; ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001; 2-tailed t test. Animal studies were performed in accordance with a protocol approved by the Kumamoto University Institutional Animal Care and Use Committee. BMT, bone marrow transplantation.

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To investigate the molecular mechanisms of hematopoietic failure after Dis3 depletion, we isolated 100 HSCs from WT and Dis3Δ/ΔMx mice and performed random displacement amplification sequencing analysis. We isolated HSCs on day 4 after the mice were administered 2 doses of pIpC on days 0 and 2, because the frequency of HSCs in Dis3Δ/ΔMx mice markedly reduced within a week after pIpC injection. Cell surface expression of Sca-1 is perturbed immediately after pIpC treatment.7 Therefore, we used the LESLAM (LineageEPCR+CD150+CD48) definition for HSC sorting (supplemental Figure 2A),8 and validated proper isolation of HSCs by confirming the expression of HSC signature genes (supplemental Figure 2B).9 Under this condition, Cre recombination was insufficient, but Dis3 expression was significantly reduced in the HSCs of Dis3Δ/ΔMx mice (∼50% reduction compared with that in WT mice; supplemental Figure 2B). Consistent with the previous finding,4,5 we observed accumulation of various noncoding RNAs in Dis3-deficient cells (supplemental Figure 2C). A total of 535 genes were upregulated, whereas 304 genes were downregulated in the HSCs of Dis3Δ/ΔMx mice compared with in HSCs from WT mice (cutoff of >1.5-fold change with false discovery rate of <0.05; Figure 2A).10 Pathway analysis revealed that genes associated with DNA damage response were enriched in upregulated genes (Figure 2B; supplemental Figure 2D). To further explore the significance of gene expression changes, we performed gene set enrichment analysis. Consistent with the previous reports that Dis3 is involved in B-cell differentiation,4,5,Dis3-deficient cells significantly increased the expression of genes that were upregulated in Lin cells compared with that in B cells (Figure 2C). Interestingly, Dis3-deficient cells also increased expression of advanced MM signature genes (Figure 2C). Furthermore, the genes related to TP53 pathway and DNA damage response were induced in Dis3-deficient cells, confirming the result of pathway analysis (Figure 2C-D). Indeed, the BM cells isolated from Dis3Δ/ΔMx mice had DNA damage, as evidenced by the elevated γH2AX (Figure 2E), in agreement with the molecular phenotype of Dis3-deficient cancer cell lines.11 Furthermore, we found an increase of cleaved caspase-3– and annexin V–positive cells in the BM of Dis3Δ/ΔMx mice (Figure 2E-F). These results indicate that loss of Dis3 induces DNA damage, leading to apoptosis of hematopoietic cells.

Figure 2.

Loss of Dis3 induces DNA damage, resulting in apoptosis of hematopoietic cells. (A) Heat map showing differentially expressed genes between the HSCs isolated from WT and Dis3Δ/ΔMx mice, 4 days after Dis3 depletion. Differentially expressed genes were selected based on >1.5-fold change with false discovery rate of <0.05. (B) Dot plot depicting top-ranked WikiPathways terms enriched in upregulated genes in the HSCs of Dis3Δ/ΔMx mice. (C) Gene set enrichment analysis plots for the indicated gene sets comparing the HSCs of WT and Dis3Δ/ΔMx mice. (D) Volcano plot showing upregulation of DNA damage checkpoint genes in the HSCs of Dis3Δ/ΔMx mice. (E) Immunoblot analysis for γH2AX and caspase-3 in the BM cells of WT and Dis3Δ/ΔMx mice, 5 days after Dis3 depletion (n = 3). Histone H3 (H3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control for each membrane. (F) Frequency of annexin V+ apoptotic cells among CD45.2+ cells in the BM of WT and Dis3Δ/ΔMx mice, 7 days after Dis3 depletion (n = 5). Representative plots of flow cytometric analysis (left panel) and summarized data (right panel) are shown. Data are mean ± standard error of the mean; ∗∗P < .01 and ∗∗∗P < .001; 2-tailed t test.

Figure 2.

Loss of Dis3 induces DNA damage, resulting in apoptosis of hematopoietic cells. (A) Heat map showing differentially expressed genes between the HSCs isolated from WT and Dis3Δ/ΔMx mice, 4 days after Dis3 depletion. Differentially expressed genes were selected based on >1.5-fold change with false discovery rate of <0.05. (B) Dot plot depicting top-ranked WikiPathways terms enriched in upregulated genes in the HSCs of Dis3Δ/ΔMx mice. (C) Gene set enrichment analysis plots for the indicated gene sets comparing the HSCs of WT and Dis3Δ/ΔMx mice. (D) Volcano plot showing upregulation of DNA damage checkpoint genes in the HSCs of Dis3Δ/ΔMx mice. (E) Immunoblot analysis for γH2AX and caspase-3 in the BM cells of WT and Dis3Δ/ΔMx mice, 5 days after Dis3 depletion (n = 3). Histone H3 (H3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control for each membrane. (F) Frequency of annexin V+ apoptotic cells among CD45.2+ cells in the BM of WT and Dis3Δ/ΔMx mice, 7 days after Dis3 depletion (n = 5). Representative plots of flow cytometric analysis (left panel) and summarized data (right panel) are shown. Data are mean ± standard error of the mean; ∗∗P < .01 and ∗∗∗P < .001; 2-tailed t test.

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Finally, we examined whether loss of Dis3 in late B cells drives MM. To do so, we crossed Dis3fl/wt or Dis3fl/fl mice with Cγ1-Cre mice in which Cre recombinase is activated in the germinal center B cells on immunization.12 We immunized 8- to 12-week-old Dis3wt/wt;Cγ1-Cre, Dis3fl/wt;Cγ1-Cre, and Dis3fl/fl;Cγ1-Cre mice with NP-chicken γ globulin (NP-CGG), and rechallenged with NP-CGG, 4 weeks after primary immunization (supplemental Figure 3A). We confirmed generation of KO allele in the BM and spleen cells isolated from Dis3fl/wt;Cγ1-Cre, and Dis3fl/fl;Cγ1-Cre mice (supplemental Figure 3B-C). In the clinical samples of patients with MM, DIS3 mutations significantly coexist with t(14;16).1 Thus, we also generated Dis3fl/wt;Cγ1-Cre;Eμ c-Maf TG and Dis3fl/fl;Cγ1-Cre;Eμ c-Maf TG mice by using t(14;16) model mice (Eμ c-Maf TG mice).13 We hereafter referred to Dis3wt/wt;Cγ1-Cre, Dis3fl/wt;Cγ1-Cre, Dis3fl/fl;Cγ1-Cre, Dis3fl/wt;Cγ1-Cre;Eμ c-Maf TG, and Dis3fl/fl;Cγ1-Cre;Eμ c-Maf TG mice immunized with NP-CGG as WT, Dis3Δ/+Cγ1, Dis3Δ/ΔCγ1, Dis3Δ/+Cγ1c-Maf, and Dis3Δ/ΔCγ1c-Maf mice, respectively (supplemental Figure 3A). During a long-term observation period, Dis3Δ/+Cγ1c-Maf and Dis3Δ/ΔCγ1c-Maf mice exhibited B-cell lymphoma (1 of 23 and 1 of 6 mice, respectively), consistent with a previous report that showed that a fraction of Eμ c-Maf TG mice developed B-cell lymphoma after a long latency.13 However, the compound mice that we generated did not exhibit an increase in the frequency of plasma cells in the BM (supplemental Figure 3D-E), and showed similar survival compared with that of WT mice (supplemental Figure 3F), indicating that these mice did not develop MM.

In conclusion, Dis3 prevents accumulation of DNA damage in hematopoietic cells, thereby supporting hematopoiesis. Furthermore, loss of Dis3 alone, and in combination with c-MAF transgene in germinal center B cells, do not exhibit plasma cell neoplasm in mice. Although recent studies have informed on the pathological significance of DIS3 deficiency in MM using in vitro experiments,11,14 it has not been determined in vivo. Our study suggests that additional oncogenic events along with DIS3 deficiency are required for myelomagenesis. Future studies await to determine the role of DIS3 in the development of MM.

Acknowledgments: The authors thank Satoru Takahashi for providing Eμ c-Maf TG mice, and Yutaka Okuno for advice.

This work was supported by grants from the Japan Society for the Promotion of Science KAKENHI (20K08734 and JP 16H06276 [AdAMS], to H.O.), the Shinnihon Foundation of Advanced Medical Treatment Research (H.O.), the Friends of Leukemia Research Fund (H.O.), the SGH Foundation (H.O.), and the Research Clusters of Tokushima University (T. Harada and H.O.).

Contribution: H.O. and Y.O. designed and performed the experiments, analyzed the data, and wrote the manuscript; S.K. performed HSC sorting; K.E., A.H., and S.U. performed random displacement amplification sequencing analysis; T.Y.-N. and J.B. assisted with bone marrow transplantation; T. Masuda, Y.K., T. Harada, M.N., T. Minami, and T. Hideshima analyzed the data; K.A. generated the Dis3-floxed mice; and G.S. designed experiments, provided resources, and edited the manuscript.

Conflict-of-interest disclosures: The authors declare no competing financial interests.

Correspondence: Hiroto Ohguchi, Division of Disease Epigenetics, Institute of Resource Development and Analysis, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan; email: ohguchi@kumamoto-u.ac.jp.

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Author notes

Random displacement amplification sequencing data have been deposited in the Gene Expression Omnibus database under accession number GSE244166.

The online version of this article contains a data supplement.

Supplemental data