Dosage-Sensitive Role of Growth Factor Independence 1 (Gfi1) In the Development of T-Cell Leukemia

Abstract 706

Gfi1 is a transcriptional repressor with important roles in the development and malignant transformation of T-cells. Gfi1 deficient mice have reduced thymic cellularity and show a partial block of differentiation of the double negative (CD4/CD8) pre-T cells into more mature stages. It is also known that Gfi1 is preferably selected as a proviral insertion site in retrovirally induced T-cell tumors in mice, which led to the concept that high-level expression of Gfi1 may be required or may drive T-cell leukemogenesis. This was consistent with findings reporting the emergence of T-cell tumors in transgenic mice over expressing Gfi1. However, lower levels of GFI1 expression was shown to be a strong indicator for worse prognosis in a group of T-ALL patients, although higher levels would have been expected. To find a solution for this discrepancy, we investigated whether Gfi1 dosage may be important in T-cell leukemogenesis or even in normal T-cell development. We have generated a new strain of knockin mice, which express a human GFI1 gene either at the same level as the endogenous wild type Gfi1 gene (Gfi1^KI/KI) or at reduced levels (Gfi1^KD/KD). We observed that low-level expression of Gfi1 leads to a reduction of thymocyte numbers (3 fold), and neutropenia in bone marrow and blood (p≤0.01). This correlates well with the phenotype of previously described Gfi1 knockout mice (Gfi1^KO/KO). Interestingly however, unlike Gfi1^KO/KO mice, Gfi1^KD/KD animals showed a normal B-cell development, suggesting that low Gfi1 levels disturb T-cell and myeloid development but are sufficient to support the differentiation of B-cells. Next we induced T-ALL in wt, Gfi1^KO/KO or Gfi1^KD/KD mice by injecting them with a single dose of N-ethyl-N-Nitroso-urea. About 15 % of wt mice developed T-cell tumors with a median latency of 140 days as was expected. Absence of Gfi1 (Gfi1^KO/KO) impeded tumor development with only 5 % of mice developing tumors and this within a longer latency period. In contrast, Gfi1^KD/KD mice developed tumors with a higher incidence (57%) and shorter latency period (100 days) than wt or Gfi1^KO/KO mice (p≤0.01 for incidence and latency). Also Gfi1^KD/KD mice developing a tumor showed a 3 fold higher number of blasts in the peripheral blood that wt mice developing T-cell leukemia (p≤0.01). Both the shorter latency period and the higher number of blasts is indicative of a more aggressive disease course in animals with low levels of Gfi1 expression. Interestingly, not only tumors arising in Gfi1^KD/KD showed lower level of Gfi1 expression but also T-ALL cells from ENU treated wt mice exhibited a 4–8 fold lower expression level of Gfi1 than normal non-leukemic wt thymocytes suggesting that Gfi1 is downregulated upon development of leukemia. Evasion of apoptosis is one hallmark of leukemia initiation and thus we studied how the different level of Gfi1 might influence apoptotic answer. We observed that Gfi1^KO/KO thymocytes were more sensitive to DNA induced cell death than wt thymocytes and underwent 2–3 times more apoptosis (p≤0.01) after DNA damage induction, which might explain why absence of Gfi1 impedes tumor initiation. In contrast Gfi1^KD/KD thymocytes showed a similar rate of apoptosis as wt thymocytes. We conclude that reduced levels of Gfi1 are not sufficient to sustain a fully normal pre-T cell development, but protect against apoptosis. Thus, the differentiation block, which is a prerequisite in any leukemia development, and a protection against apoptosis might accelerate T-cell leukemia development in Gfi1^KD/KD mice. This would indicate that the role of Gfi1 in the malignant transformation of T-cell is dose dependent and that low level of Gfi1 expression (but not absence) favors leukemia development and this might be one explanation why lower level of Gfi1 is associated with worse prognosis in T-ALL patients.