A Murine Model Presenting Leukemic Transformation from MPN with a Mutation in Epigenetic Modifier
Authors: Sato Yuka , Ueda Koki , Minakawa Keiji , Miura Saori , Ikeda Kazuhiko 
Published: December 2024

Myeloproliferative neoplasms (MPNs) are chronic disease with increased blood cell counts and various complications. Progression to myelofibrosis (MF) or acute myeloid leukemia (AML) from chronic MPNs is one of the most life-threatening events. MPNs arise from hematopoietic cells harboring driver mutations such as JAK2-V617F and flame shifts of CALR. Additional genetic alterations such as mutations in epigenetic modifiers, DNMT3A, TET2, ASXL1 and EZH2 make impacts on disease phenotypes. However, whether these mutations are associated with leukemic transformation from MPNs has been controversial. To answer this question, various murine models with double mutant of Jak2-V617F(Jak2VF) and epigenetic modifiers (Dnmt3a-/-, Tet2KD/KD, Asxl1+/-, Ezh2-/-) have been generated, however, leukemic transformation has been detected only in very limited number of mice while most of the double mutant mice presented more severe phenotypes of MPNs such as MF and extramedullary hematopoiesis compared with mice with a single mutation. We hypothesized short survival of Jak2VF mice due to severe MPN phenotypes makes it difficult to study about leukemic transformation. Recently, we generated Calr type2-like mutant mice (Calr10d/+) which shows a milder phenotype of MPN than Jak2VF mice such as mild splenomegaly, slight elevation of platelet counts and vulnerability to cardiovascular disease (J Hematol Oncol.14: 52,2021). We asked if double mutant mice with Calr10d/+ and an epigenetic modifier, Ezh2-/- or Dnmt3a-/-, allow us observation of longer duration to develop leukemic transformation compared with Jak2 mutant models.

We first generated Calr10d/+;Ezh2fl/fl;Ert-Cre-Tg mice and deleted Ezh2 by tamoxifen injection during 6 to 8-week-old to induce double knock-in (Calr10d/+;Ezh2-/-). At 10 months after deletion of Ezh2, both Calr10d/+;Ezh2-/- and Ezh2-/- presented myeloid biased hematopoiesis in peripheral blood and bone marrow compared to Calr10d/+ alone, but no overt myelofibrosis and leukemic transformation were observed. However, most of Calr10d/+;Ezh2-/- mice died with severe MF-like or AML-like disease from 12 to 16-month-old while majority of Ezh2-/- and Calr10d/+ mice survived at least 20 months. Both MF-like and AML-like moribund mice presented severe anemia and progression of splenomegaly. AML-like mice showed higher white blood cell counts compared with MF-like mice and proliferative hematopoietic stem cell fraction; Flk2-/CD48+/CD150+/Lineage-Sca-1+c-Kit+ (LSK). Moreover, secondary transplantation of AML-like bone marrow cells results in rapid reproduction of the disease and death of recipient mice, while transplantation of MF-like cells did not kill recipients in 6 months. Interestingly, bone marrow thin section specimen of AML mice did not present myelofibrosis, suggesting that MF and AML arise from different clones although it warrants further study. We have also generated Dnmt3aR878H/+ mice with driver mutations which are under observation.

To clarify the molecular mechanisms of disease progression of Calr10d/+;Ezh2-/- mice, we first performed RNA sequencing of LSK of preleukemic 10-month-old Calr10d/+;Ezh2-/- mice, and Calr10d/+ mice. Although LSK of Calr10d/+ mice showed upregulated JAK-STAT pathway compared to wildtype LSK (FDR 0.11), LSK of Calr10d/+;Ezh2-/- mice presented further overexpression of specific JAK-STAT target genes such as Il7r and Dntt compared with LSK of Calr10d/+. Upregulation of Il7r in LSK of Jak2VF;Ezh2-/- mice were also reported by us (Blood Advances.1:1001-1015,2017) and others (Exp Hematol.213:1459-1477,2016), therefore it might play important role in disease progression of MPN although canonical function of Il7r is the role in lymphoid differentiation. Also, S100 family signaling pathway was upregulated in Calr10d/+;Ezh2-/- LSK. Presently, we are analyzing LSK of MF-like and AML-like mice.

In conclusion, we generated a murine model harboring mutations in MPN driver and epigenetic modifiers, and observed not only progression of MPN phenotypes but also recurrent leukemic transformation. The molecular mechanisms are under investigation.