Abstract
Background: The WT1 gene encodes for a zinc finger-containing transcription factor involved in differentiation, cell cycle regulation and apoptosis. WT1 expression is developmentally regulated and tissue-specific, with expression maintained in the kidney and in CD34+ hematopoietic progenitor cells. WT1 mutations are reported in approximately 10-15% of both adult and pediatric patients with acute myeloid leukemia (AML), and have been associated with treatment failure and a poor prognosis. Reported mutations consist of insertions, deletions or point mutations; and occur primarily in exon 7 or exon 9 of the WT1 gene. These mutations are thought to alter WT1 DNA-binding ability or result in a loss of function. Despite these observations, the functional contribution of WT1 mutations in leukemogenesis is still largely undetermined.
Results and Methods: We have shown that transduction and expression of wild type WT1 in murine 32D cells enhances granulocytic differentiation upon treatment with G-CSF, and that expression of mutant WT1 inhibits this effect. To investigate this in a human AML cell model, we transduced U937 cells with the same WT1 vectors. Strikingly, shortly after transduction, U937 cells expressing wild type WT1 spontaneously differentiate towards a mature monocytic phenotype, but U937 cells expressing mutant WT1 do not differentiate and maintain an immature phenotype (Fig A). This relative block in U937 differentiation with mutant WT1 expression was overcome with differentiation-inducing treatment with all-trans retinoic acid (ATRA). These results suggest that mutant WT1 alters the ability of myeloid cells to terminally differentiate.
We obtained a novel knock-in WT1 mutant (WT1mut) mouse model that is heterozygous for the missense mutation R394W in exon 9, and homologous to exon 9 mutations observed in human AML. We evaluated cohorts of two-month old mice and noted an expansion of lineage negative cells and various progenitor cell compartments; particularly, the megakaryocyte-erythroid progenitor (MEP) compartment; in WT1mut bone marrow (BM) relative to wild type. We also found that lineage negative WT1mut BM cells from two-month old mice show higher in vitro colony-forming capacity and an increased ability to serially replate in methylcellulose culture compared to wild type BM cells. Flow cytometry of WT1mut cells at tertiary replating revealed an immature, largely c-kit+ population, suggesting an aberrantly enhanced self-renewal capability of myeloid progenitors in WT1mut mice.
Furthermore, survival analysis of the WT1mut mice demonstrates inferior survival compared to wild type, and several WT1mut mice were found to have anemia and myelodysplasia. To address the possibility of germ line WT1mut syndromes causing renal failure and anemia, and thereby influencing survival, we transplanted BM from each genotype into lethally irradiated congenic mice. Upon engraftment with donor marrow, the expression of WT1mut is confined to the hematopoietic system in this model. The Kaplan-Meier survival curve, based on absolute age of the BM, shows statistically significant decreased survival of WT1mut BM transplant recipients compared to wild type BM recipients (Fig B). Anemia and dysplasia were also seen in these WT1mut BM recipients; findings that are suggestive of dysfunctional hematopoiesis, and may be secondary to the changes in progenitor cell self-renewal and differentiation we have observed.
Conclusions: Leukemogenic WT1 mutations confer enhanced self-renewal of hematopoietic progenitor cells and a block in terminal myeloid differentiation in vitro, which could potentially prime cells for leukemic transformation upon acquisition of cooperative events. Mice with WT1 mutant bone marrow develop anemia and evidence of myelodysplasia, which may contribute to their decreased survival. 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.
(A) U937 cells expressing wild type WT1 spontaneously differentiate, demonstrated here by gain of monocytic markers CD11a and CD11b as measured by flow cytometry, whereas cells expressing mutant WT1 vectors 101 and 126 remain undifferentiated. (B) Mice transplanted with WT1mut bone marrow have inferior survival compared to mice transplanted with wild type bone marrow.
(A) U937 cells expressing wild type WT1 spontaneously differentiate, demonstrated here by gain of monocytic markers CD11a and CD11b as measured by flow cytometry, whereas cells expressing mutant WT1 vectors 101 and 126 remain undifferentiated. (B) Mice transplanted with WT1mut bone marrow have inferior survival compared to mice transplanted with wild type bone marrow.
No relevant conflicts of interest to declare.
