Leukemogenic WT1 Mutations Increase Proliferation by Accelerating Cell Entry Into S-Phase, and Synergize with FLT3/ITD Mutations to Enhance These Aberrant Cell Cycle Effects

Abstract

Abstract 2437 Introduction/Background:Wilms tumor 1 (WT1) gene encodes for a zinc finger-containing transcription factor thought to play a role in differentiation, cell cycle regulation and apoptosis. WT1 expression is developmentally regulated and tissue-specific, with adult expression maintained in kidney cells and early CD34+ hematopoietic progenitor cells. Inactivating mutations of this tumor suppressor gene are well described in sporadic Wilms tumor and as germline mutations in WAGR and Denys-Drash syndromes. Approximately 10% of adult and pediatric patients with cytogenetically normal acute myeloid leukemia (CN-AML) harbor WT1 mutations. Some studies suggest that patients with WT1 mutations may have a worse overall prognosis, particularly in combination with other poor prognostic indicators, such as FLT3ITD mutations. Interestingly, WT1 and FLT3ITD mutations are commonly found together, suggesting they may cooperate to cause AML. Despite these clinical observations, the functional contribution of WT1 mutations to leukemogenesis, both alone and in cooperation with FLT3ITD, is still largely undetermined. Methods/Results:Ba/F3 cells were stably transfected with the WT1 gene, including 3 different engineered mutated vectors: two missense point mutations (WT1mut101 and WT1mut146) and one nonsense insertional mutation (WT1mut126), which produces a truncated protein. These mutations have been described in pediatric patients with acute leukemia. The expression of each WT1 vector was confirmed by PCR and cDNA sequencing. We compared the proliferative rate of WT1 wildtype (WT1wt) to WT1mutated (WT1mut) transfected cells using trypan blue cell counting, and saw an early increased proliferative rate for high-expressing WT1mut compared to WT1wt and WT1 empty vector (EV). To further investigate these differences, we performed cell cycle analysis with propidium iodide (PI) staining. After synchronizing all cell lines by arresting cells in G0-G1 phase, the cells were seeded at equal concentrations and assayed for cell cycle changes at various time points. Interestingly, all WT1mut cell lines consistently showed earlier entry into S phase and therefore a decreased G1/S ratio at 24 hrs after synchronization, compared to EV and WT1wt (Fig A). This suggests that the “check-point” controlling entry into S phase is altered in cells expressing WT1 mutations, which manifests as a proliferative advantage in these cells. [Display omitted] Next, we stably transfected Ba/F3 cells with a FLT3ITD construct, and co-transfected additional cells with both WT1mut and FLT3ITD constructs. All cells were synchronized by arresting in G0-G1 phase, achieved with 24 hrs of serum and cytokine starvation and treatment with 20nM of CEP-701 (ensuring G0-G1 arrest of cells with FLT3 vectors). The cells were then washed and re-suspended in serum and cytokine-containing media, and seeded at equal concentrations. We observed that FLT3ITD cells and WT1mut126 cells had decreased and nearly equivalent G1/S ratios at early time points compared to EV and WT1wt cells, conferring similar early proliferative advantages for cells with these mutations. Interestingly, we found that cells co-transfected with both mutations enhanced this effect: the absolute number of WT1mut126+FLT3ITD cells undergoing transition into S phase was increased and this effect was seen at an earlier time point compared to WT1mut126, FLT3ITD, WT1wt or EV cells (Fig B). Conclusions:The functional and contributory role of WT1 mutations in leukemogenesis has yet to be characterized. Our preliminary in vitro data with a cell line transfected with WT1 vectors suggests that cell cycle regulation, and therefore proliferation, is aberrant in cells expressing mutated WT1. Consistent with previous reports, our data reaffirms a proliferative advantage in cells transfected with FLT3ITD mutations. We showed that these cells transition to S phase at an early time point, conferring an early proliferative advantage, and do so at an equivalent rate and time to WT1mut cells. In addition, we found that cells co-transfected with both WT1mut126+FLT3ITD demonstrated an earlier and more pronounced entry into S phases compared to either individual mutation alone. These observations deserve further investigation, as they may help explain how mutated WT1 contributes to the initiation and progression of leukemia, and how WT1 and FLT3ITD mutations may cooperate in leukemogenesis. Disclosures:No relevant conflicts of interest to declare.