Pediatric mixed phenotype acute leukemia (MPAL) is a rare, high-risk leukemia that accounts for 2-3% of pediatric leukemia cases and has a particularly poor prognosis (<50% survival). This is primarily due to lack of tailored therapies that target the unique genetic and developmental basis of this disease, highlighting a critical need to better understand MPAL biology. We recently discovered that the WT1 gene is mutated in nearly half of all pediatric T/myeloid MPAL patients, with most mutations causing premature termination codons that remove the C-terminal DNA binding domain of the WT1 protein. This particular alteration is predicted to have functional consequences, as WT1 encodes a DNA binding transcription factor important for early hematopoietic development. Furthermore, WT1 plays important roles in the regulation of gene expression, chromatin structure, and DNA methylation through interactions with other molecular complexes, highlighting the multiple pathways that may be disrupted in WT1-mutant hematopoietic cells. Importantly, each of these pathways represent potential vulnerabilities that can be exploited or directly targeted with rationally-designed therapy. The research proposed in this Fellowship will identify the developmental and molecular consequences of WT1 mutations in early hematopoiesis, providing a critical first step towards developing new strategies to combat this difficult to treat childhood disease.
Aim 1 of this research proposal will address the role of truncating WT1 mutations in perturbing early hematopoiesis. A CRISPR/Cas9-based approach will be used to introduce patient-relevant mutations at the endogenous WT1 locus in human hematopoietic stem and progenitor cells (HSPCs) which will faithfully model the genetic and developmental origin of T/myeloid MPAL. Wild-type and WT1-mutant cells will be differentiated in vitro using colony forming assays and in vivo through transplantation into humanized mice. Flow cytometry and gene expression profiling will identify distinct hematopoietic populations and gene expression programs that are disrupted by WT1 alterations.
Aim 2 of this research proposal will use proteomics and functional genomics assays to identify the molecular alterations induced by truncating WT1 mutations in human HSPCs, complementing the developmental alterations identified in Aim 1. Specifically, these assays will map dynamic protein-protein interactions, chromatin modifications, and DNA methylation patterns in wild-type and WT1-mutant HSPCs. Integration of these data will enable identification of the molecular mechanisms underlying transcriptional changes that deregulate early hematopoiesis in WT1-mutant MPAL. Overall, these studies will elucidate the developmental and molecular consequences of recurrent WT1 alterations in T/myeloid MPAL and reveal new pathways and dependencies that may be interrogated for therapeutic benefit.
Despite high cure rates for many pediatric cancers, high-risk leukemias including mixed phenotype acute leukemia (MPAL) remain challenging to cure due to lack of tailored therapies that target the genetic and developmental basis of these diseases. The proposed research plan will interrogate the mechanism of action of the commonly mutated WT1 gene in pediatric MPAL using a combination of human cellular models and high- throughput genomics and proteomics methodologies. This research plan will contribute to our basic understanding of the early stages of MPAL development and has the potential to identify new molecular targets that can be interrogated for therapeutic intervention.
