Xist RNA is a potent suppressor of hematologic cancer in mice

Abstract

X chromosome aneuploidies have long been associated with human cancers, but causality has not been established. In mammals, X chromosome inactivation (XCI) is triggered by Xist RNA to equalize gene expression between the sexes. Here we delete Xist in the blood compartment of mice and demonstrate that mutant females develop a highly aggressive myeloproliferative neoplasm and myelodysplastic syndrome (mixed MPN/MDS) with 100% penetrance. Significant disease components include primary myelofibrosis, leukemia, histiocytic sarcoma, and vasculitis. Xist-deficient hematopoietic stem cells (HSCs) show aberrant maturation and age-dependent loss. Reconstitution experiments indicate that MPN/MDS and myelofibrosis are of hematopoietic rather than stromal origin. We propose that Xist loss results in X reactivation and consequent genome-wide changes that lead to cancer, thereby causally linking the X chromosome to cancer in mice. Thus, Xist RNA not only is required to maintain XCI but also suppresses cancer in vivo.

Copyright © 2013 Elsevier Inc. All rights reserved.

Figures

Figure 1. Deleting Xist in the Blood Compartment Results in Female Specific Lethality

(A) Map of the Xist2lox and XistΔ alleles and FISH probes used to distinguish the two alleles. Xist RNA is detected by using (Cy5-Sx9 probe, cyan). Representative RNA/DNA FISH are shown (n ≥ 50 each). Δ, deletion. X, XbaI site. (B) Xist RNA FISH of splenocytes (n ≥ 100). Xist RNA: green, FITC-Sx9 probe. (C) Female specific lethality: Kaplan-Meier kill curves plotted over 750 days were generated using Prism (GraphPad software). There were no differences between any control group: Vav-Cre, Xist2lox/+, or Xist2lox/Xist2lox females or corresponding male controls. These control genotypes were combined: females, “ Xist2lox/+”; males, “Xist2lox/Y”. (D) Female-specific splenomegaly. Top panels: representative mutant females and age-matched controls are shown. Note abdominal swelling in 27-1-1 (XistΔ/+, 6.3 months old at death) and cervical mass in 8-1-1 (XistΔ/XistΔ, 7 months old at death). Animal ID numbers are shown with genotype throughout the figures. Bottom: representative spleens from each male and female genotype are shown. (E) Age-dependent increase in the spleen-to-body weight ratio. Ratios taken at 2 (n = 2–6), 4 (n = 6), and 5 to 16 (n = 14–36) months of age. Means ± SEM are shown. Significance of the differences between mutant and WT spleen were calculated using the Student’s t test. **p ≤ 0.01; ***p ≤ 0.001. (F) Temporal progression in spleen pathology. Immunostains are as indicated. Scale bars, 100 µm. See also Figures S1 and S2.

Figure 2. Bone Marrow Insufficiency

(A) Reticulin staining of bone sections reveals progressive hypocellularity and myelofibrosis (black stains representing reticulin fibers) in mutant females. XistΔ/+ cases are shown. Scale bars, 50 µm. (B) Representative bone marrow cytology from WT and mutant females, with anomalies indicated. All were Wright-Giemsa stained except siderocytes stained by Prussian blue to reveal pathological presence of nonhemoglobin iron. (C) Quantitation of bone marrow cells in seven mutant (Mut1–Mut7) and four WT (WT1–3 and 7) females in tabular (top) and histogram (bottom) form. M:E, myeloid to erythroid ratio. Mac, macrophages. Means ± SEM are shown. **p ≤ 0.01; *p ≤ 0.05. (D) Nonphysiological EMH in multiple organs (17-1-4, XistΔ/XistΔ, 6 months old at death; 8-1-1, XistΔ/XistΔ, 7 months old at death). All sections were H&E stained. Scale bars, 50 µm.

Figure 3. Multilineage Dysplasia and Myeloproliferative Neoplasia

(A) Representative peripheral blood smears from WT and mutant females, Wright-Giemsa stained unless noted. (B) Hematologic analysis of end-stage mutants and age-matched control.

Figure 4. Histiocytic Sarcoma and Lymphoplasmacytic Vasculitis

(A) Enlarged lymph nodes from end-stage mutants shown with a WT control. Cervical lymph nodes of 27-1-1 (XistΔ/+, 6.3 months old) and brachial lymph node of 31-2-1 (XistΔ/+, 5.8 months old) are shown. (B) Top: sections of enlarged lymph node from 21-3-1 (XistΔ/+, 8.8 months old at death) with follicular B and T cell and intra- and interfollicular histiocytic expansion are shown. Bottom: sections of enlarged lymph node from 8-1-1 (XistΔ/XistΔ, 7 months old at death) with a plasmacytoma-like infiltrate are shown. Lymphoplasmacytic vasculitis is also shown. See also Figure S2O. (C) Masses containing metastatic histiocytic sarcoma (arrows) in end-stage liver and kidney. (D) H&E stains of metastatic histiocytic sarcoma in multiple organs. Scale bars represent 100 µm. (E) Immunohistochemistry confirms histiocytic sarcoma. Scale bars represent 100 µm. (F) Histiocytic sarcoma in bone marrow. Scale bars represent 100 µm. Note positive F4/80 staining for intralesional giant cells (inset).

Figure 5. Transplantability of MPN/MDS Suggests a Hematopoietic Rather Than Stromal Origin

(A) Schematic of transplantation experiments. (B) Dysplasia in peripheral blood and bone marrow of mutant-to-WT transplants. See also Figures S3A–S3C. (C) Histiocytic sarcoma recapitulated in WT recipients of mutant bone marrow or splenocytes. Recipient livers and spleen (1434) were pale from anemia. White masses in recipient spleen contain histiocytic sarcoma (arrow). Mice succumbed within 40 days after transplantation. (D) H&E stain of EMH in matched donor and recipient livers and spleens, as indicated. (E) Immunohistochemistry confirms histiocytic sarcoma in donor and recipient (middle panels). Reticulin stain of bone marrow shows exuberant myelofibrosis in donor (at necropsy) and recipient (40 days after transplantation). (F) WT-to-mutant transplantations reversed disease. WT-to-WT transplantation controls were normal. Scale bars represent 100 µm. See also Figure S4.

Figure 6. A Primary Defect in the HSC

(A) Representative FACS analysis of bone marrow cells from a 5-month-old female mutant and a WT littermate mouse showing increased LSK+/LSK− ratio and decreased number of SLAM-enriched long-term HSCs (LSK+CD48−CD150+) in mutant mice. (B) Histograms of bone marrow LSK+/LSK− ratios and SLAM-enriched long-term HSCs suggest, respectively, a failure of maturation and loss of progenitors in mutant (n = 12) female mice in comparison to WT female mice (n = 13). Means ± SEM shown; Student’s t test, ***p ≤ 0.001. (C) Progressive loss of SLAM-enriched long-term HSCs in bone marrow of mutant mice (n = 12) over time as compared to WT mice (n = 13). R2= 0.5317. (D and E) Competitive repopulation assays reveal maturation defects in mutant cells. WT (Xist2lox/+) or mutant (XistΔ/+) cells were mixed at 1:1 ratio with cells isolated from congenic WT Ly-5.1+ mice and transplanted into WT hybrid Ly-5.1 +/Ly-5.2+ recipients (n = 4–5 per group). Peripheral blood was sampled for FACS analysis repeatedly for 2.5 months (D), and bone marrow was sampled at 2.5 months (E). Means ± are SEM shown; *p < 0.05, **p ≤ 0.01. (F) FACS analysis of bone marrow cells in 4-month-old mutant and WT mice (n = 4 mice per group). Means ± SEM shown; *p +/LSK− ratio and a decreased number of SLAM-enriched long-term HSCs in mutant mice. (H) Histograms of splenic LSK+/LSK− ratios and SLAM-enriched long-term HSCs suggest, respectively, a failure of maturation and loss of progenitors over time in mutant females (n = 4) as compared to WT females (n = 6). Means ± SEM shown. Student’s t tests show *p ≤ 0.05 and **p ≤ 0.01. (I) Progressive loss of SLAM-enriched long-term HSCs in spleen of mutant mice (n = 4) over time as compared to WT mice (n = 6). R2 = 0.846. (J) FACS analyses show that HSC maturation defects (elevated LSK+/LSK− ratios) and loss of SLAM-enriched long-term HSCs are recapitulated in the WT recipients of mutant bone marrow cells at 2 months post-Tp. (K) FACS analyses of reverse transplantation (WT-to-mutant), with WT-to-WT control. At 2 months post-Tp with WT bone marrow cells, mutant 39-1-2 (XistΔ/+, 8.2 months) shows a correction of LSK+/LSK− ratio and number of SLAM-enriched long-term HSCs (right panels), in comparison to pre-Tp profiles (left panels). See also Figures S3, S5, and S6.

Figure 7. Gene Expression Profiling by Microarray Analysis Reveals X Reactivation and Genome-wide Changes

(A) List of upregulated X-linked transcripts in blood cells of Xist mutants. See Figure S1A for details. (B) Hierarchical clustering of transcripts that are differentially expressed between WT and mutant blood cells, as listed in Tables S1B,S1C, and S1D. Enriched functional categories are indicated on left. See also Figure S7 and Tables S1A–S1I. (C) A model for the pathogenesis and progression of cancer resulting from Xist loss.