A Wilms Tumor 1 (WT1) Mutation Causes Myelodysplastic Syndrome in a Knock-in Mouse Model, and a Mixed Myelodysplastic/Myeloproliferative Neoplam in Double Knock-in Mice with WT1 and FLT3/ITD Mutations

Abstract

Background: WT1 is a zinc finger transcriptional regulator and acts as a tumor suppressor gene in various cell types. WT1 mutations are reported in approximately 10% of both adult and pediatric patients with acute myeloid leukemia (AML), and at a lower frequency in patients with myelodysplastic syndome (MDS). Reported mutations consist of insertions, deletions or point mutations, and are thought to alter WT1 DNA-binding ability and result in a loss of function. WT1 mutations are associated with FLT3/ITD mutations in AML, suggesting possible leukemogenic cooperativity, and yet WT1 mutations have been independently associated with treatment failure and a poor prognosis. Recently, a physical interaction demonstrated between WT1 and TET2 suggests a common functional pathway, and explains the mutual exclusivity of these mutations in AML. Despite these observations, the functional contribution of WT1 mutations in hematologic malignancies is not entirely understood. To our knowledge, we are the first to describe here a hematologic phenotype in a WT1 mutant mouse model and in a novel WT1 mutant x FLT3/ITD crossbred mouse model.Methods: Knock-in WT1 mutant mice are heterozygous for missense mutation R394W in the DNA-binding domain, which has been described in cases of human AML. Mice with a heterozygous 18-bp ITD knocked into the FLT3 gene were crossbred with the WT1 mutant mice, and Kaplan-Meier survival analysis was performed across genotypes. CBCs and BM cytospin morphology from moribund mutant mice were compared to wild type controls. To create a transplant model, 2e6 whole BM cells from each genotype were injected into lethally irradiated congenic mice. Competitive transplants were performed by injecting a 1:1 ratio of CD45.1 wild type (control) cells with CD45.2 WT1 mutant or wild type (test) cells into lethally irradiated C45.1 recipients.Results: We noted an expansion of lineage negative cells and various progenitor cell compartments in WT1 mutant (WT1mut) BM relative to wild type (wt); including the megakaryocyte-erythroid progenitor (MEP) compartment. WT1mut BM cells from two-month old mice showed an increased ability to serially replate in methylcellulose culture compared to wt BM cells, demonstrating aberrantly enhanced self-renewal capacity.WT1mut mice demonstrated a trend towards an inferior late survival compared to wt in survival analysis, and several moribund WT1mut mice were found to have anemia and erythrodysplasia. Most ITD mice developed a fatal myeloproliferative neoplasm (MPN), as previously described. Interestingly, double mutant mice (WT1mut+ITD) had an inferior survival compared to ITD (p <0.001) or WT1mut alone, and BM from moribund WT1mut+ITD mice demonstrated features of both erythrodysplasia and myeloproliferation, diagnostic of MDS/MPN. We then transplanted BM from each genotype into lethally irradiated congenic mice. WT1mut BM transplant recipients showed a trend toward inferior survival compared to wt BM recipients. Strikingly, all evaluable WT1mut recipient mice developed MDS in this model; manifested as decreased hemoglobin (p = 0.03), clear erythrodysplasia, and a decreased myeloid:erythroid (M:E) ratio. ITD BM recipients developed MPN as expected, and the majority of WT1mut+ITD BM recipients developed MDS/MPN. Finally, in a competitive transplant model, WT1mut BM cells exhibited an initial engraftment disadvantage, followed by a increasing engraftment advantage over wt BM cells at later time points - further supportive data that WT1mut BM causes dysfunctional hematopoiesis, a hallmark of MDS.Conclusion: BM cells from mice with the leukemogenic WT1 mutation R394W demonstrate enhanced self-renewal of hematopoietic progenitor cells, which could potentially prime cells for leukemic transformation upon acquisition of cooperative events. Importantly, transplanted mice with WT1 mutant BM consistently develop MDS, manifested as anemia and erythrodysplasia and contributing to a trend in decreased survival. Mice with both this WT1 mutation and a FLT3/ITD mutation develop a mixed MDS/MPN phenotype, which is a discrete diagnostic entity, and results in a more aggressive disease and inferior survival to mice with ITD mutations alone. These data provide new and important insights into the aberrant functional effects of WT1 mutations on hematopoiesis, and are the first to characterize the hematopoietic phenotype of a WT1 mutation in vivo. DisclosuresNo relevant conflicts of interest to declare.