2016 David F. Silbert Summer Fellowship Research

Fellow

Summer research project abstract
WT1 loss-of-function mutations in Acute Myeloid Leukemia
Gang M; Christopher MJ; Ley TJ

Introduction: Mutations in Wilms tumor 1 (WT1) were recently identified in 11 of 42 patients with Acute Promyelocytic Leukemia (APL), a morphologic subtype of Acute Myeloid Leukemia (AML). WT1 mutations may therefore cooperate with promyelocytic leukemia retinoic acid receptor alpha (PML-RARA) in APL development. Paradoxically, wild type WT1 is expressed highly in APL and AML subtypes compared to normal hematopoietic progenitors and high levels of WT1 expression are associated with poor outcomes. It is therefore unclear whether WT1 plays a role in promoting leukemic progression or functions as a tumor suppressor in hematopoietic cells.

Methods: cDNAs coding for wild type WT1 and a truncated WT1 protein (mimicking common AML mutations) were cloned into retroviruses to allow overexpression in mouse bone marrow expressing the PML-RARA fusion protein. The effect of WT1 overexpression was then analyzed by flow cytometry to measure the frequency of GFP-tagged populations. To examine the effects of the loss of WT1 in human cells, a lentivirus encoding the CRISPR guide RNA, Cas9 enzyme, and GFP tag was used to inactivate WT1 in AML-derived cell lines. Constructs were generated, one targeting the 5 end to generate a nonsense mutation and another targeting the 3 end to closely mimic the current model of AML mutations. The effect of truncation or DNA-binding domain mutations in WT1 was also quantified by flow cytometry and protein expression was measured by western blotting.

Results: Bone marrow isolated from PML-RARA mutant mice overexpressing full length WT1 showed a decrease in aberrant self-renewal of hematopoietic progenitor cells. In contrast, overexpression of truncated WT1 protein showed no significant loss of self-renewal in bone marrow progenitors from mutant mice. Experiments will extend these studies to PML-RARA-transformed hematopoietic progenitor cells. In addition, we optimized a CRISPR-CAS9 system to disrupt WT1 in two human AML-derived cell lines that express WT1. Future experiments will test whether CRISPR/Cas9-mediated loss of WT1 expression leads to changes in cell growth of primary human AML cells and AML-derived cell lines.

Conclusions: Our initial findings suggest that overexpression of wild type WT1 prevents self-renewal in pre-leukemic PML-RARA-mutant mouse hematopoietic progenitors. This raises the possibility that WT1 mutations arising in AML increase the ability of leukemic cells to proliferate. Further research will be directed towards understanding how wild type WT1 is regulated in AML and whether CRISPR/Cas9-induced mutations in wild type WT1 leads to an enhanced proliferation in primary human AML cells.