Overcoming Therapy Resistance in Fusion Oncoprotein Driven Pediatric Leukemia

PROJECT SUMMARY Leukemia represents the most prevalent form of cancer among children. Sequencing studies from identical twins indicate that the initial genetic alterations can manifest as early as during fetal development. Acute myeloid leukemia (AML) is a heterogeneous disease, which is characterized by various genetic alterations

including fusion oncoproteins. NUP98-NSD1 fusion-positive AML is a poor prognostic subtype that is commonly diagnosed in pediatric patients with cytogenetically normal AML. There, the nuclear pore complex member NUP98 fuses to the histone methyltransferase NSD1. Recently, NUP98-NSD1 gene fusions, in combination with

transcription factor WT1 (Wilms' tumor 1) mutations, were identified in a chemotherapy resistant pediatric AML cohort, with a devastating 4-year event-free survival of less than 10%. The underlying reason why NUP98-NSD1 with WT1 mutations are associated with therapy resistance and relapse is not known.

In preliminary experiments, we created a humanized system of NUP98-NSD1 rearranged leukemia with endogenous expression of the fusion gene, with and without WT1 loss of function mutations. Strikingly, NUP98- NSD1 fusion-driven leukemogenesis displayed a developmental dependency, where its expression transforms

pre-natal fetal liver-derived hematopoietic stem cells (HSCs), to a lesser extent post-natal cord blood-derived HSCs, but unable to transform adult bone marrow-derived HSCs. In vitro, endogenous NUP98-NSD1 oncoprotein conferred clonal selection and proliferation advantage in fetal HSCs, while in vivo, through

xenotransplantation in mice, NUP98-NSD1 faithfully recapitulated AML. Therefore, we hypothesize that the epigenetic state of fetal-derived HSCs is permissive of NUP98-NSD1-mediated leukemic transformation, but is repressed in developmental states after birth. The overall objective of our project is to define mechanisms of action of NUP98-NSD1 in HSCs across

development, understand how WT1 mutations drive therapy resistance and identify novel therapeutic targets for NUP98-rearranged leukemia. In Aim 1, we are proposing to investigate the cellular and developmental dependency of NUP98-NSD1-mediated leukemic initiation by xenotransplantations. We will assess the single-

cell epigenetic and transcriptional landscape of NUP98-NSD1 fusion-positive HSCs across ontogeny. In Aim 2, we plan to characterize the downstream effects of WT1 mutations in NUP98-rearranged leukemia. We observed that concurrent WT1 loss led to a more primitive stem cell hierarchy and an enrichment of quiescent leukemic

stem cells. Finally, in Aim 3, we are proposing to identify therapeutic approaches against NUP98-rearranged leukemia. We will functionally assess the role of candidate genes using our established model and primary PDX patient samples. Our goal is to characterize novel therapeutic targets for NUP98-rearranged leukemia.

The proposed research has the potential to change our understanding of why children are susceptible to NUP98-NSD1 rearranged AML and provide a strong rational for therapeutic intervention in the future.