BAALC Regulates Hematopoietic Stem Cells Through P53 and Promotes Leukemia with Impaired P53 Function

Abstract 48

Elevated expression of BAALC (brain and acute leukemia, cytoplasmic) is an adverse prognostic factor in patients with cytogenetically normal acute myeloid leukemia (CNAML). However, its precise role in normal hematopoiesis and leukemogenesis remains to be elucidated. To address this issue, we evaluated the effect of BAALC overexpression upon hematopoiesis and leukemogenesis and generated BAALC-deficient mice.

First, we examined the effect of BAALC overexpression upon hematopoiesis. BAALC-transduced bone marrow (BM) cells had decreased colony-forming capacity. Bone marrow transplantation (BMT) assay demonstrated that the donor chimerism was lower in transplanted mice with BAALC-overexpressed cells than with control cells. To examine the mechanism, we performed gene expression analysis. This revealed that p53 pathway is up-regulated in BAALC-overexpressed cells. Colony-forming assay and BMT assay using p53-deficient BM cells demonstrated that the blockage of proliferation by BAALC overexpression is canceled in p53-deficient cells in vitro and in vivo, suggesting that the inhibition of proliferation by BAALC overexpression is dependent on p53 pathway.

Next, we generated BAALC-conventionally deficient mice. Most BAALC-deficient female mice died between E11.5 and E12.5 because of defects of placental development. BAALC-deficient E11.5 yolk sac cells had increased colony-forming capacity. Transplantation assay showed that the donor chimerism at 4 weeks after transplantation was higher in transplanted mice with BAALC-deficient E12.5 fetal liver cells than with wild fetal liver cells. Moreover, the frequency of G0/G1 phase cells was decreased in BAALC-deficient yolk sac. Thus, the proliferation is activated and the frequency of quiescent cells is reduced in BAALC-deficient embryonic hematopoietic cells. In addition, we examined the role of BAALC in adult hematopoiesis using BAALC-conditionally deficient mice with Mx-Cre system. In BAALC-deficient (Mx-Cre⁺BAALC flox/flox) BM, long-term hematopoietic stem cells (HSCs), identified as CD34⁻Flt3⁻cKit⁺Sca1⁺Lin⁻ cells, were reduced as compared with control (Mx-Cre⁻BAALC flox/flox) BM. BAALC-deficient adult BM had impaired long-term repopulating ability in serial competitive BMT. Moreover, BAALC-deficient mice showed higher mortality rates after weekly 5-FU injections. On the other hand, the ability of colony forming unit spleen (CFU-S) was enhanced and the recovery after myelosuppression induced by single 5-FU injection was faster in BAALC-deficient mice, indicating that BAALC-deficient HSCs have enhanced short-term repopulating ability. Next, to address the relationship between BAALC and p53 protein levels, we performed intracellular flow cytometric analysis and immunocytochemical staining. These experiments revealed that p53 protein is reduced in BAALC-deficient HSCs. Immunoprecipitation revealed that BAALC interferes with the binding of MDM2 to p53, which leads to stabilization of p53 protein.

Finally, we tested the effect of BAALC overexpression in leukemia mouse models. BAALC overexpression promoted leukemia in mice in cooperation with TEL/PDGFBR-AML1/ETO (TPAE). BM cells transduced with TPAE and BAALC had increased colony-forming capacity as compared with TPAE and mock-transduced cells. By contrast, BAALC cannot accelerate myc/BCL2-induced leukemia. Western blot showed that p53 is inactivated in TPAE leukemic cells but not in myc/BCL2 leukemic cells. BM cells transduced with myc/BCL2 and BAALC had decreased colony-forming capacity as compared with myc/BCL2 and mock-transduced cells. However, BAALC overexpression promoted colony-forming capacity of p53-deficient cells transduced with myc/BCL2. Similarly, BAALC overexpression promoted the colony-forming capacity of BM cells transduced with BCR/ABL which is known to inactivate p53. Promoted proliferation of BCR/ABL-transduced cells by BAALC overexpression was canceled by the MDM2 antagonist, Nutlin-3. Taken together, BAALC overexpression accelerates leukemia with impaired p53 pathway.

In conclusion, these data suggest that BAALC activates p53 pathway and maintains the stemness in normal HSCs. Once p53 pathway is impaired, however, BAALC promotes proliferation of leukemic cells. Our data for the first time uncovered a novel function of BAALC, which may lead to novel therapeutic strategy for CNAML with poor prognosis.