Experimental and preclinical modeling of NUP98-rearranged acute leukemia

Project Summary The overarching goals of the original FusOnC2 consortium award titled Experimental and Preclinical Modeling of NUP98-rearranged Acute Leukemia are to understand the mechanistic basis of NUP98 FO-driven leukemogenesis and to develop and translate new, more effective therapeutic approaches for this form of

leukemia. Work in Project 1 of this consortium seeks to develop and characterize more faithful experimental models of NUP98-rearranged leukemia and leverage these models to improve our understanding of the biology and potential therapeutic targeting of this high-risk subset of acute leukemias. In this application, we propose

the use of integrated cell barcoding and multi-omic single-cell sequencing in mouse and human models of NUP98-rearranged leukemia to dissect how NUP98 FO, cell-of-origin, and transcriptional dysregulation intersect to drive leukemogenesis and leukemia cell phenotype. The proposed research is complimentary to but not

overlapping with our currently funded projects. Additionally, it will provide interactive collaborations with Drs. Shalin Naik and Mark Dawson, as well as with members of their research groups, who have previously used lineage tracing to study hematopoietic development and malignancy. To achieve the goals of this complimentary

supplemental application, we propose the following specific aims: Aim 1: To identify the gene expression profile, chromatin accessibility landscape, and immunophenotype of mouse HSPCs permissive to NUP98 FO-driven leukemia. Here, we will obtain single-cell profiling and lineage tracing (SPLINTR) barcoding libraries from our collaborators, and optimize their use in mouse hematopoietic

stem and progenitor cells (HSPCs). We will then leverage penetrant mouse models of NUP98::KDM5A and NUP98::LNP1 fusion oncoproteins (FOs), which have been developed in the Mullighan and Klco laboratories, and combine SPLINTR barcodes with empty vector, NUP98::KDM5A, or NUP98::LNP1 FO. FO+barcode+ cells

will be profiled under in vitro conditions as well as after transplantation and leukemia development in vivo. Samples will be subjected to multi-omic single-cell profiling (DOGMA-seq) before and after the introduction of FO to identify changes in gene expression, chromatin accessibility, and immunophenotype driven by NUP98

rearrangement. Furthermore, single-cell sequencing of bone marrow and spleen samples from leukemic mice will allow us to assess the gene regulatory state and stem cell/progenitor subtypes permissive to leukemogenesis in vivo. Aim 2: To characterize the differentiation and gene regulation patterns induced by NUP98 FOs in conditions

suitable for multilineage human hematopoiesis. In this aim, we will apply SPLINTR barcoding to human models of NUP98 rearrangement. SPLINTR barcodes will be introduced in cord blood CD34+ HSPCs, which will subsequently be transduced to express empty vector, NUP98::KDM5A, or NUP98::LNP1. Cells will be exposed

to in vitro conditions suitable for multilineage hematopoiesis, and changes in gene expression, chromatin accessibility, and immunophenotype will be determined via DOGMA-seq to identify the effect of FO expression on gene regulation and differentiation. Furthermore, we will perform similar studies followed by transplantation

of FO+barcode+ CD34+ cells into immunodeficient mice. These studies will provide insights on the FO-induced changes that occur in vivo and compare them with the results we observe in in vitro, in mouse models (Aim 1), and in NUP98-rearranged patient samples.