Egr1 and Apc Haploinsufficiency Cooperate in the Pathogenesis of Myeloid Neoplasia: A Mouse Model for Therapy-Related Myeloid Neoplasms with a Del(5q)

Abstract 117

Heterozygous deletions of the long arm of chromosome 5 are among the most common abnormalities in de novo (∼15% of patients) and therapy-related myeloid neoplasms (t-MN) (∼40% of patients). Two minimally deleted segments have been identified - the minimally deleted segment within 5q31.2 is associated with de novo AML and t-MNs, whereas the other spans 5q33.1 and is associated with MDS with an isolated del(5q). Current studies support a haploinsufficiency model, in which loss of a single allele of more than one gene on 5q contributes to the development of myeloid neoplasms. Using mouse models, we previously showed that haploinsufficiency of Egr1 (5q31.2) or Apc (5q22-frequently deleted in t-MN) independently recapitulates some features of human myelodysplastic syndromes (MDS).

To test the hypothesis that reduced levels of EGR1 and APC cooperate in the pathogenesis of MDS/AML, we generated mice expressing a single allele of Egr1 and Apc: Mx1-Cre⁺Apc^fl/+Egr1^+/−(Apc^del/+Egr1^+/−). At 2 mos of age, we induced deletion of a single allele of Apc by injection of 3 doses of pI-pC. Survival curves clearly show that Egr1 and Apc haploinsufficiency cooperate in the development of disease with a median survival of 129 days for Apc^del/+Egr1^+/− mice and 296 days for Apc^del/+mice (P<0.0001). Although disease latency was significantly shorter for Apc^del/+Egr1^+/− mice, their phenotype was similar to Apc^del/+ mice, with only two exceptions. For both cohorts, mice typically developed splenomegaly and a lethal macrocytic anemia with monocytosis. Anemic mice had an increased proportion of CD71⁺Ter119⁺ erythroblasts, indicating a block in erythroid development between the early and late basophilic erythroblast stage. Two mice displayed anemia and leukocytosis (WBC 51–72 k/mL) with an increased proportion of Mac1⁺ cells in the spleen and Kit⁺ cells in the bone marrow (1 mouse). As anticipated, mice with wild type levels of Apc (Mx1-Cre^-Apc^fl/+) or with loss of one allele of Egr1 showed no signs of anemia.

Mutations in TP53 are commonly found in t-MNs with a del(5q) and loss of Tp53 in mouse models has been shown to promote AML by enabling aberrant self renewal. To test the hypothesis that loss of TP53 may adversely advance disease development, we crossed Tp53^+/− to Egr1^+/− and Apc^del/+ mice. Similar to Apc^del/+Egr1^+/− mice, Apc^del/+Tp53^+/− mice rapidly developed macrocytic anemia with a median survival of 144 days, suggesting that partial loss of TP53 function accelerates the Apc^del/+ -induced macrocytic anemia. Triple heterozygous mice (Apc^del/+Tp53^+/−Egr1^+/−) had a median survival of 178 days, but survival was not statistically different than Apc^del/+Egr1^+/− mice (P=0.35) suggesting that Egr1 and Tp53 loss play redundant roles in the development of disease in Apc^del/+ mice. Thus, in the context of Apc haploinsufficiency, loss of Egr1 or Tp53 function promotes erythroid failure. These results are in contrast to the setting of ribosomal protein haploinsufficiency, as is the case in MDS with an isolated del(5q), where induction of TP53 is essential for erythroid failure.

It has been proposed that inactivation of TP53 (through additional TP53 mutations) would be required for progression to AML, in the setting of a 5q deletion. To this end we transduced Egr1^+/−Apc^del/+ bone marrow cells with a Tp53-specific shRNA, known to reduce Tp53 transcripts by ∼90%, and transplanted them into lethally irradiated C57BL/6 mice. Although penetrance of disease was low, 2 out of 13 mice (15%) developed an aggressive AML, as compared to 0 of 12 mice transplanted with Egr1^+/−Apc^del/+ cells transduced with control shRNA. These data suggest that EGR1 and APC haploinsufficiency cooperate in the development of myeloid disorders, characterized by ineffective erythropoiesis, and that further mutations, such as that achieved by complete inactivation of TP53, are required for progression to AML.