Knockout of HMGA1 Promotes ATRA-Induced Differentiation in MLL-Rearranged Leukemia

Introduction: HMGA1 is a small non-histone chromatin protein characterized by 3 AT-hooks, which modulates transcription by altering the chromatin architecture. It tends to be highly expressed in poorly differentiated cells including hematopoietic stem cells. In various kinds of solid tumors, HMGA1 has been reported to play as an oncogene, and its upregulation was reported to be associated with poorer clinical outcomes. However, although recent study demonstrated that HMGA1 was associated with progression of JAK2^V617F positive myeloproliferative neoplasms to myelofibrosis or leukemia, the roles of HMGA1 in myeloid malignancies were still unclear. Therefore, we analyzed its function in myeloid malignancies using HMGA1 knockout (KO) mice. We could not detect clear differences in peripheral blood counts or bone marrow cells, and generated various kinds of models of acute myeloid leukemia (AML). Knockout of HMGA1 could not delay the onset of these leukemia, but we could demonstrate that leukemic cells with MLL-ENL fusion gene which lacked in HMGA1 gene were more susceptible to all-trans retinoic acid (ATRA)-induced expression of CD11b, which suggested that HMGA1 could be associated with the differentiation process induced by ATRA.

Method: We generated Vav-iCre HMGA1^flox/flox mice, and we first analyzed the fraction of peripheral blood cells and bone marrow cells of these mice using flow cytometry. Next, to evaluate roles of HMGA1 in AML, we made various murine models of AML. We collected c-kit positive fractions of bone marrow cells from the KO mice or Vav-iCre(-) HMGA1^flox/flox mice and retrovirally introduced MLL-ENL, MOZ-TIF2, BCL2-MYC fusions. Primary transplantation was performed to sublethally irradiated (5Gy) mice with 4.0×10⁵ infected cells for each fusion gene. Secondary transplantation was also performed for MLL-ENL and BCL2-MYC with 1.0×10⁴ cells for each. Blood samples were collected weekly, and disease-free survivals were compared by Kaplan-Meier method with Vav-iCre(-) HMGA1^flox/flox mice. Also, leukemic cells infected with MLL-ENL were collected, and these cells were cultured for four days with different concentrations (1μM, 5μM or 10μM) of ATRA. Flow cytometry analysis was performed to evaluate the differentiation induced by ATRA with antibodies to murine CD11b, c-kit and Gr-1.

Result: There was no significant difference in birth rate between Vav-iCre(+) and Vav-iCre(-) mice, which suggested that HMGA1 was not essential for fetal hematopoiesis. Peripheral blood cell counts were measured at week 8 and week 20, and fractions of peripheral blood and bone marrow cells were assessed at week 8 using flow cytometry, but no significant differences were observed. As for AML models, there were no significant differences in survival periods after primary or secondary transplantation. Leukemic cells retrovirally induced with MLL-ENL were collected from primary recipients, and these cells were treated with ATRA. Although growth rates were not show significantly different, expression levels of CD11b after exposure to ATRA were significantly higher in Vav-Cre (+) mice (5μM: 70% vs 44%, respectively; p=0.035. 10μM: 80% vs 46%, respectively; p=0.0006) as shown in the following figure.

Discussion: We established HMGA1 KO mice and assessed the function of HMGA1 both in normal and leukemic hemopoiesis. Although HMGA1 might have little effect on normal hematogenesis, it seemed that HMGA1 had inhibitory effect on ATRA-induced differentiation process of MLL-ENL leukemic cells. Though more conditions including AML types and differentiation methods should be tested and the molecular mechanisms should be scrutinized, there is a possibility that inhibition of HMGA1 is effective in combination with differentiation treatments.

Masamoto:Otsuka Pharmaceutical Co., Ltd: Honoraria; ONO PHARMACEUTICAL CO., LTD: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Chugai Pharmaceutical Company: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria, Research Funding; Nippon Shinyaku Co., Ltd: Honoraria; AbbVie GK: Honoraria; Janssen Pharmaceutical K.K: Honoraria; Bristol Myers Squibb: Honoraria; Asahi Kasei Pharma: Honoraria; Sanofi: Honoraria; Yamasa Corporation: Honoraria; AstraZeneca: Honoraria; SymBio Pharmaceuticals: Honoraria; MSD K.K: Honoraria. Kurokawa:Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Kyowa Kirin Co., Ltd.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company .: Research Funding, Speakers Bureau; AbbVie GK: Research Funding; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; SANWA KAGAKU KENKYUSHO CO., LTD.: Consultancy, Speakers Bureau; Astellas Pharma Inc.、Eisai Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Asahi Kasei Pharma Corporation.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; AstraZeneca K.K.: Speakers Bureau; SymBio Pharmaceuticals Limited.: Speakers Bureau; Amgen Inc.: Speakers Bureau; Sanofi K.K.: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau.