Runx1 deficiency predisposes mice to T-lymphoblastic lymphoma

Abstract

Chromosomal rearrangements affecting RUNX1 and CBFB are common in acute leukemias. These mutations result in the expression of fusion proteins that act dominant-negatively to suppress the normal function of the Runt-related transcription factor 1 (RUNX)/core binding factor beta (CBFbeta) complexes. In addition, loss-of-function mutations in Runt-related transcription factor 1 (RUNX1) have been identified in sporadic cases of acute myeloid leukemia (AML) and in association with the familial platelet disorder with propensity to develop AML (FPD/AML). In order to examine the hypothesis that decreased gene dosage of RUNX1 may be a critical event in the development of leukemia, we treated chimeric mice generated from Runx1(lacZ/lacZ) embryonic stem (ES) cells that have homozygous disruption of the Runx1 gene with N-ethyl-N-nitrosourea (ENU). We observed an increased incidence of T-lymphoblastic lymphoma in Runx1(lacZ/lacZ) compared with wild-type chimeras and confirmed that the tumors were of ES-cell origin. Our results therefore suggest that deficiency of Runx1 can indeed predispose mice to hematopoietic malignancies.

Figure 1.

Runx1^lacZ/lacZ chimeras are more susceptible to developing T-lymphoblastic lymphoma than wild-type chimeras. (A) Kaplan-Meier survival curves (lymphoma-free) are shown for untreated Runx1^lacZ/lacZ (n = 6) and Runx1^lacZ/lacZ (n = 6) and wild-type (n = 6) chimeras treated with 100 mg/kg ENU. • indicates untreated Runx1^lacZ/lacZ chimeras; ▪, wild-type ES-cell chimeras treated with ENU; ▴, Runx1^lacZ/lacZ chimeras treated with ENU. (B) Hematoxylin and eosin (H&E) staining of thymus, bone marrow (BM), and liver from LZD nos. 2 through 5. Immunohistochemistry using an anti-CD3 antibody demonstrates that the malignant cells in the livers of these animals stained positively for CD3. (C) Flow cytometry of thymocytes from LZD no. 2 and LZD no. 4 using antibodies against CD4 and CD8 demonstrates that the majority of cells have an abnormal CD4⁺/CD8⁺ phenotype. WT indicates wild type. Numbers in each quadrant indicate the percentage of total thymocytes in that quadrant.

Figure 2.

T-lymphoblastic lymphoma is of ES-cell origin in Runx1^lacZ/lacZ chimeras. (A) Immunohistochemistry using an anti–β-galactosidase antibody demonstrates that the malignant cells in the spleens of LZD nos. 2 through 5 express β-galactosidase and are therefore derived from Runx1^lacZ/lacZ ES cells. The normal splenocytes from LZD no. 6 (which did not develop lymphoma) serve as negative controls. (B) Southern blot analysis detects presence of only the Runx1-LacZ knock-in allele in lymphoma-effaced bone marrow from LZD nos. 2 through 4 but only wild-type Runx1 allele from normal bone marrow of LZD no. 6. (C) Genomic PCR using LacZ gene primers and DNA from sorted thymic cells of a Runx1^lacZ/lacZ chimera. Lane 1, CD8⁺; lane 2, CD8⁺/CD4⁺; lane 3, CD4^-/CD8^-; lane 4, CD4⁺; lane 5, total thymus; lane 6, total thymus from a wild-type mouse; lane 7, no DNA. (D) Genomic PCR using LacZ gene primers and DNA from sorted bone marrow cells of a Runx1^lacZ/lacZ chimera. Lane 1, c-kit⁺/lin^-; lane 2, c-kit⁺/lin⁺; lane 3, c-kit^-/lin⁺; lane 4, c-kit^-/lin^-; lane 5, total bone marrow.