RALDH2 Is Regulated by an LMO/SCL(TAL1) Associated Protein Complex in T Cell Acute Lymphoblastic Leukemia.

Three and five years after an otherwise successful gene therapy trial for X1-SCID (gamma c deficiency) three out of 11 patients developed T cell leukemia due to insertional activation of the proto-oncogene LMO2 after retroviral mediated gamma c transfer into autologous CD34+ cells. In one of the patient’s T-cells SCL(TAL1) was expressed in addition to LMO2 because of a SIL-SCL fusion, where the deletion of the SIL gene brings SCL(TAL1) under the control of the constitutively active SIL promoter. LMO2 and SCL(TAL1) are components of a multifactorial transcription regulation complex consisting of SCLTAL1, LMO2, GATA1/2, Ldb-1, and E2A which is essential for normal hematopoiesis; however, LMO2 and SCL(TAL1) are normally down regulated after the DN2 stage of T cell maturation and expression of both proteins after DN2 leads to a block of T cell differentiation which precedes the onset of malignancy as shown in transgeneic mouse models. Experiments with T cell lymphoblasts of the patient, who developed LMO2/SCL(TAL1) associated T-ALL after gene therapy were initiated, in order to determine genes which are deregulated by aberrant expression of LMO2/SCL(TAL1) in mature T-cells leading to the onset of T-ALL. We established a xenotransplant model in NOD/SCID mice with the patient’s leukemic T-cells, which will serve as an in vivo model to examine the mechanisms underlying LMO2/SCL(TAL1) associated T-ALL. By immuno precipitation using LMO2 specific antibodies and nuclear extracts of the patient’s leukemic T-cells, we were able to isolate a protein complex containing LMO2, SCL(TAL) and E47 which resembles the LMO1 associated protein complex described by Ono et al. for the T-ALL cell line Jurkat. Gene transcription profiling using whole genome gene chips (Affymetrix) revealed, that in addition to LMO2 and TAL1, RALDH2 (retinaldehyde dehydrogenase) were among genes which were aberrantly expressed in the patient 5 T-cell clone, whereas in normal T-cell controls none of the three genes are transcribed. Immunoblot analyses and RT-PCR revealed an N-terminal truncation of RALDH2 (T-RALDH2) in the patient’s T-ALL cells. A cellular based activity test using protein extracts of the patient’s T-cells, K562 cells (positive control) and HL-60 cells (negative control) revealed, that the truncated form of RALDH2 is enzymatically active and converts retinaldehyde into retinoic acid which was described to induce cell proliferation and to inhibit activation induced apoptosis of T-cells. To test the role and regulation of RALDH2 in T-ALL we designed RALDH2 and LMO1 specific siRNA for down regulation of RALHD2 and LMO1 in a T-ALL cell culture model (Jurkat cells). We found that down regulation of LMO1 led to a decrease of RALDH2 expression whereas SCL(TAL1) and house keeping genes were not affected. This result confirms the finding of Ono et al. who showed that LMO and SCL(TAL1) activate a truncated form of RALDH2. Both, down regulation of LMO1 or RALDH2 resulted in a decrease of cell viability between 24 and 48 hours post siRNA transfection. We conclude that RALDH2 is a target gene of an LMO1(2)/SCL(TAL1) associated transcription regulating complex in T-ALL and might play a crucial role in the onset of TALL by interfering with proliferation and apoptotic processes.