BCL2A1 Is a Survival and Immortalization Factor for Primitive Hematopoietic Cells.

Abstract 2957

Poster Board II-933

We recently reported the development of an acute myeloid leukemia in a rhesus macaque transplanted with autologous CD34+ cells transduced with a murine stem cell virus-derived replication defective retrovirus vector expressing only marker genes under control of the strong MCSV LTR. This animal had an unusual clonal reconstitution pattern the first year following transplant, with a single transduced myeloid progenitor cell clone accounting for up to 80% of then normal myelopoiesis (Kelly, 2003). The same vector-containing clone then transformed to AML five years following transplantation, and each tumor cell was shown to contain two vector insertions, one localized 20 kb upstream the CDw92 gene on chromosome 9, and the second localized in the first intron of BCL2A1 on chromosome 15 (Seggewiss, 2006), a gene belonging to the anti-apoptotic BCL2 family not previously linked to myeloid leukemia. BCL2A1 was highly expressed in the tumor cells. This tumor was the first hematopoietic malignancy reported in a recipient of primitive cells transduced with a replication-incompetent vector containing only marker genes, and suggested that BCL2A1 could have potent effects on hematopoiesis. To further investigate the impact of the BCL2A1 gene product on normal and malignant hematopoiesis, we cloned the murine and human HA-tagged BCL2A1 cDNAs into lentiviral vectors and transduced the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these proteins in vitro. We confirmed the role of BCL2A1 as an anti-apoptotic protein. The proliferation and survival of the BaF3 cells is dependant on IL-3. Upon removal of IL-3 from the media, BaF3 cells underwent an arrest in the G1 phase of the cycle. Untransduced cells or cells transduced with the empty lentiviral vector were 45% apoptotic, but this fraction decreased to 30 and 15% respectively with the cells transduced with murine and human BCL2A1. Similar results were obtained in murine 32Dcl3 cells and human UT7/Epo-S1 cells, two cells line that are respectively dependant on IL-3 and erythropoietin. In order to study the in vivo impact of BCL2A1 on hematopoiesis, C57/bl6 (Ly5.2) mice have been transplanted with primary bone marrow cells (from C57/bl6 (Ly5.1)) transduced with the BCL2A1 and control vectors, and have been followed for in vivo expansion of transduced clones and development of leukemia. The mice cohort consisted of 5 MOCK controls, 15 control vector expressing GFP only, and 15 murine BCL2A1. We frequently checked the blood counts and analyzed the lineage of blood by FACS. Mice transplanted with marrow cells transduced with the BCL2A1 vector had higher overall marking levels in the blood compared to the vector control (80% vs. 10% cells GFP+ respectively). Interestingly, preliminary histopathology revealed that 7 mice from the BCL2A1 group, but none of the control groups developed a further to be classified very poorly differentiated hematologic malignancy consisting of circulating blasts, splenomegaly, and lymphadenopathy. Cells responsible for this fatal disease were not stained by markers used for FACS analysis or by immunohistochemistry. The median overall survival was significantly different for mice treated with the vector (420 days) and mice treated with BCL2A1 (328 days). The median disease free survival was more striking as the median could not be defined for the vector while it was of 397 days for BCL2A1. Two hundred and fifty three days after the transplantation we selected 3 primary mice from BCL2A1 and vector control groups to perform secondary transplant. Less than a month after reinfusion all BCL2A1 mice developed a disease characterized by high white blood cell counts with a majority of undifferenciated cells, as well as splenomegaly, and hepatomegaly. These results were repeated in a second set of secondary transplant carried out 289 days after transplantation confirming that the disease is transplantable. The preliminary histopathology findings showed a similar phenotype to the lymphatic malignancy seen in the primary mice. In conclusion, we have confirmed the anti-apoptotic role of BCL2A1, and we are now investigating its role in hematopoiesis and leukemogenesis. We are investigating the exact phenotype of the disease seen in primary and secondary mice in order to clarify the role of BCL2A1 as a survival factor.