Figure 1.

Alkylating agent therapy increases the incidence of myeloid neoplasms. A, Schematic of ENU treatment and transplantation scheme used to develop a mouse model of t-MN. Egr1^+/−, Apc^del/+ BM cells were transduced with Trp53 shRNA, and transplanted into lethally irradiated WT recipient mice. For the ENU cohort, donor mice were treated once with 100 mg/kg ENU 2 weeks before BM harvest; recipients were treated once with 100 mg/kg ENU, 2 to 3 weeks before lethal irradiation and transplantation. B, Kaplan–Meier survival curves of untreated and ENU-treated mice. Percentage survival (time to sacrifice) is plotted versus time in days. In the absence of ENU treatment, the mice survive significantly longer (508 days vs. 200 days, P < 0.001), with a small percentage developing MDS/AML, but the majority succumb to advanced age and nonhematologic effects. C, Histologic classification of diseases that arose in the no-ENU and ENU-treated mice. There is a significantly increased frequency of AML in the ENU-treated group. Most of the no-ENU mice died due to age-related issues rather than hematopoietic malignancies. Disease frequency was compared using Fisher exact test. D, Images of the myeloid disease in ENU-treated mice were obtained using an Olympus BX41 microscope and a 50×/0.9 (oil) or 40×/0.9 objective, and processed with Adobe Photoshop. Peripheral blood smears, BM smears, and spleen touch preparations were stained with Wright–Giemsa (500× magnification), and spleen sections were stained with hematoxylin and eosin (200× magnification). Examples of a myeloblast (inset i), dysplastic granulocyte (inset ii), sheets of infiltrating blasts (inset iii), and a dysplastic erythroid precursor (inset iv) are shown. E and F, LSK⁺ (Lin-, Sca1⁺, Kit⁺) cells were sorted from three different mouse cohorts: recipients of WT, luc shRNA⁺ BM (controls; n = 3), recipients of Egr1^+/−, Apc^del/+, Trp53 shRNA⁺ cells (EA-Trp53), either untreated (n = 3) or treated with ENU (n = 3), approximately 70 to 90 days after transplant, prior to the onset of overt leukemia. E, GSEA of WT control (n = 3) compared with EA-Trp53 LSK⁺ samples [includes both the no-ENU and ENU-treated groups (n = 6)] to identify premalignant changes as a consequence of Egr1, Apc, and Trp53 loss. F, GSEA of EA-Trp53 LSK⁺ samples from the no-ENU (n = 3) versus ENU-treated (n = 3) group was used to identify genetic consequences of in vivo ENU exposure. Biological pathways/processes that were significantly enriched (FDR<20%, nominal P < 0.05) in two or more Molecular Signatures Database (MSigDB) gene sets are shown. Heatmap of the normalized enrichment scores (NES) shows that DDR pathways (DNA repair, apoptosis, checkpoints) are downregulated due to loss of Egr1, Apc, and Trp53, with or without ENU treatment. E, Energy production pathways, such as mTORC1, protein translation, and glycolysis, are upregulated as a consequence of ENU exposure (F).