Cell intrinsic and extrinsic factors synergize in mice with haploinsufficiency for Tp53, and two human del(5q) genes, Egr1 and Apc

Abstract

Therapy-related myeloid neoplasms (t-MN) are a late complication of the successful use of cytotoxic therapy for patients with cancer. A heterozygous deletions of the long arm of chromosome 5 [del(5q)], observed in 40% of patients, is associated with prior exposure to alkylating agents, and a high frequency of TP53 loss or mutation. In previous studies, we demonstrated that haploinsufficiency of 2 del(5q) genes, Egr1, and Apc, individually play a role in the pathogenesis of hematologic disease in mice. We now show that loss of one copy of Egr1 or Tp53 in an Apc haploinsufficient background (Apc (del/+)) accelerated the development of a macrocytic anemia with monocytosis, early features of t-MN. The development of anemia was significantly accelerated by treatment of mice with the alkylating agent, N-ethyl-N-nitrosourea (ENU), regardless of the levels of expression of Egr1 and Tp53. Transplantation of either wild type; Egr1(+/-); Tp53(+/-); Apc(del/+); or Egr1(+/-), Apc(del/+) bone marrow cells into lethally irradiated Apc(del/+) recipients resulted in rapid development of anemia that was further accelerated by administration of ENU to recipients, demonstrating that the Apc(del/+)-induced anemia was cell extrinsic and potentiated by ENU mutagenesis. These data emphasize the synergistic role of cell intrinsic and cell extrinsic (microenvironment) factors in the pathogenesis of t-MN, and raise awareness of the deleterious effects of cytotoxic therapy on the stromal microenvironment.

Figure 1

Loss of 1 Allele of Egr1, Tp53, or both genes equally accelerates Apc ^del/+-induced fatal anemia. Kaplan-Meier survival curve of Apc^fl/+ control mice and Apc^del/+mice, crossed with Egr1^+/− (A), Tp53^+/− (B), or Egr1^+/− and Tp53^+/− (C). Percent survival (time to sacrifice of moribund animals) is plotted vs time in days, post-pIpC treatment (to induce Apc deletion). The number of mice in each cohort is shown. Egr1^+/−, Apc^del/+; Tp53^+/−, Apc^del/+; and Egr1^+/−, Tp53^+/−, Apc^del/+ mice all had significantly decreased survival compared with Apc^del/+ mice (P < .0001). The deletion of a single allele of Apc was induced in Mx1-Cre⁺Apc^fl/+ mice treated with 3 doses of pI-pC, referred to as Apc^del/+mice. Control Mx1-Cre⁻Apc^fl/+ mice are referred as Apc^fl/+mice.

Figure 2

All mice with Apc haploinsufficiency develop a macrocytic anemia with monocytosis. (A) CBC of Apc^del/+and Apc^fl/+mice alone or crossed with Egr1^+/−(E), or Tp53^+/− (P), or Egr1^+/−, Tp53^+/− (EP) are shown. CBCs of Apc^del/+ mice were taken at the time of sacrifice, when the mice were severely anemic and moribund. Blood counts of Apc^fl/+ control mice were taken during a similar timeframe, but mice were not moribund. (B) Representative peripheral blood smears (×1000) and spleen sections at ×40 and ×500 from anemic Apc^del/+mice alone or crossed and of Apc^fl/+ control mice. Images were obtained using an Olympus microscope (Model BX51), equipped with an Optronics 3CCD 1080p digital camera, and processed with Adobe Photoshop. MO, monocytes; NE, neutrophils; PLT, platelets; WBC, white blood cells.

Figure 3

Anemia in all Apc haploinsufficient mice is caused by a block at an early erythroblast stage. (A) Splenocytes were isolated from Apc^fl/+ control mice, Apc^del/+ mice, or Apc^del/+mice crossed with Egr1^+/−(E), Tp53^+/− (P), or Egr1^+/−, Tp53^+/− (EP), and flow cytometric analysis of immature erythroblasts (CD71⁺Ter119⁺), mature erythroid cells (CD71⁻Ter119⁺), granulocytes (Gr1⁺Mac1⁺), B cells (CD19⁺), and T cells (CD4⁺ or CD8⁺) was performed when mice displayed severe anemia. (B) Flow cytometric analysis of spleen cells from a representative, anemic Apc haploinsufficient mouse. The 4r erythroid cell populations from least to most differentiated are indicated as R1 to R4. R1, pro-erythroblasts; R2, basophilic erythroblasts; R3, polychromatophilic erythroblasts; R4, orthochromatophilic erythroblasts.

Figure 4

Anemia and block in erythropoiesis is detected earlier in Egr1^+/−, Apc^del/+ double heterozygous mice compared with Apc^del/+ mice. (A) Peripheral blood counts of Apc^fl/+, Apc^del/+, and Apc^del/+, Egr1^+/− mice 3 months after induction. (B) Flow cytometric analysis of spleen cells isolated from mice, 3 months postinduction. Ter119^high cells were analyzed using the CD71 and FSC parameters. Ery.A (Ter119^highCD71^highFSC^high) are basophilic, and Ery.B (Ter119^highCD71^highFSC^low) are late basophilic and polychromatic, whereas Ery.C (Ter119^highCD71^lowFSC^low) are orthochromatic erythroblasts and reticulocytes. A representative plot is shown on the right. Data represents the relative erythroid populations and standard error of the mean for 3 mice for each genotype. (C) The frequency of apoptosis in the R2 (CD71⁺Ter119⁺) population was measured by Annexin V staining. A representative contour plot is shown on the right. Apc^del/+ and Egr1^+/−, Apc^del/+ mice consistently displayed a trend toward less apoptosis in the CD71⁺Ter119⁺ population compared with controls. For all experiments, the average and standard error of the mean of 3 mice per genotype is shown. HCT, hemocrit.

Figure 5

ENU-treated mice with Apc haploinsufficiency develop a fatal macrocytic anemia at an accelerated pace, regardless of Egr1 or Tp53 expression. (A) Kaplan-Meier survival curve of Apc^del/+mice, or mice crossed with Egr1^+/−; Tp53^+/−; or Egr1^+/− and Tp53^+/−. Mice were treated with 3 doses of pI-pC at 2 months of age, and ENU, where indicated, at 3 months. All mice treated with ENU died at a significantly faster rate (P < .0001). (B) CBC are shown for ENU-treated Apc^fl/+and Apc^del/+mice or Apc^del/+mice crossed with Egr1^+/−(E), Tp53^+/− (P), or Egr1^+/−, Tp53^+/− (EP). CBCs of Apc^del/+ mice were taken at the time of sacrifice, when the mice were severely anemic and moribund. Blood counts of ENU-treated Apc^fl/+ control mice were taken during a similar timeframe, but mice were not moribund. MO, monocytes; PLT, platelets; WBC, white blood cells.

Figure 6

The fatal anemia is induced by an Apc-haploinsufficient BM microenvironment (A) Kaplan-Meier survival curve of Apc^fl/+ (WT) or Apc^del/+ recipients transplanted with WT, Egr1^+/; Tp53^+/−; Apc^del/+; or Egr1^+/−, Apc^del/+BM cells. Median survival for Apc^del/+recipients was 130, 118, 139, 130, and 139 days, respectively. All Apc^fl/+and Apc^del/+mice were treated with pIpC at 2 months, and recipients were lethally irradiated and transplanted 4 weeks postinduction. (B) Kaplan-Meier survival curve of Apc^fl/+ (WT) or Apc^del/+ recipients transplanted with Apc-WT (CD45.1/CD45.2) BM cells. A representative dot plot shows that Apc^del/+ BM are CD45.2⁺, whereas Apc^del/+ mice are reconstituted with CD45.1⁺ CD45.2⁺ BM cells. (C) PCR analysis of splenocytes isolated from Apc^del/+ recipients transplanted with WT or Egr1^+/− BM cells. Analysis of Egr1 and Apc clearly indicates that splenocytes are donor-derived. A PCR control on the right indicates expected sizes of deleted and WT bands. (D) Analysis of deletion of Apc, as determined by PCR analysis of DNA from stromal cells: Apc^fl/fl (Control), Apc^del/del (Apc-null), Apc^del/+(Apc-het), and WT. In the sample shown, a PCR product from the floxed Apc allele was still visible in Apc^del/del and Apc^del/+ samples, in addition to the anticipated deleted and WT bands, suggesting that deletion in stromal cells may not always occur in every cell. This is in contrast to hematopoietic cells, where the deletion occurs in all cells. (E) Western blot analysis of extracts from spleen cells and stromal cell cultures using β-catenin antibody and actin as a loading control. Apc deletion had occurred in virtually all cells in the stromal cells used for this analysis (not shown).

Figure 7

The fatal anemia is slightly accelerated by pretreatment of Apc^del/+ recipients with ENU. (A) A model showing that Apc^fl/+and Apc^del/+ recipients (CD45.2⁺) were treated with pIpC at 2 months, ENU at 2.5 months (where indicated), and transplanted 4 weeks post-pIpC treatment with CD45.1/CD45.2 WT BM. (B) Kaplan-Meier survival curve of Apc^fl/+ (WT) or Apc^del/+ recipients transplanted with CD45.1/CD45.2 WT BM. Recipients treated with ENU are indicated (dashed line). (C) CBC of untreated and ENU-treated Apc^fl/+and Apc^del/+recipient mice. RBC, Hb, and MCV were significantly different for both untreated and ENU-treated Apc^del/+recipient mice compared with Apc^fl/+-recipient controls. Monocytosis was significant only for the ENU-treated Apc^del/+ vs Apc^fl/+-recipient mice. MO, monocytes.