Abstract 3924: Cooperation between mutant IL7R and TLX3 generates mixed myeloid lymphoid leukemia from thymocyte progenitors
Authors: Rodrigues Gisele 
Published: April 2023

Abstract
Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite the current pediatric treatment protocols have resulted in 5-year overall survival rates of up to 90%, treatments have substantial side effects. Therefore, it is important to identify more specific therapeutic targets and develop more effective and less toxic drugs and drug combinations. We have shown that overexpression of TLX3 in association with mutant IL7R (mutIL7R) is sufficient to generate aggressive leukemia from transduced CD4-CD8- (DN) thymocyte progenitors. To better understand mutIL7R/TLX3-mouse leukemia, we combined single cell RNA sequencing (scRNAseq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), DNA Next Generation Sequencing (NGS) Chromatin Immunoprecipitation Sequencing (ChIP-Seq) and flow cytometry to explore the cooperation of mutIL7R and TLX3 in orchestrating leukemogenesis. We found that mutIL-7R+TLX3 induced murine mixed T/myeloid leukemia. Myeloid cell clones (CD11b+) expressed identical TCR rearrangements as the T cell clones (CD8+), indicating that mutant IL-7R+TLX3 generated mixed leukemia from T cell progenitors. Moreover, the passaged leukemia lost the T-cell phenotype and acquired a pro-B-like phenotype, suggesting preferential expansion of pro-B cells (CD19+, B220+ IgM-), which were not detected in the founder leukemia presumably due to its low frequency. We next sought to determine whether the oncogene combination is sufficient to drive a similar immunophenotype profile in vitro. We showed that ectopic expression of mutIL7R and TLX3 in DN primary thymocytes growing on OP9-DL4 pushed the cells to myeloid (CD11b+, Gr1+) and B-cell (B220) phenotype over a time course of 4, 8 and 20 days, while T-cell development was blocked, suggesting that TLX3 expression prevented differentiation into CD4+/CD8+ T-cells. To better understand the genes regulated by TLX3 on driving a mixed phenotype leukemia we performed ChiP-seq of D1 cell line ectopically expressing mutIL7R and TLX3. We identified RUNX as a possible key mediator of the mixed-phenotype-leukemia induced by TLX3 in combination with mutIL7R. Additionally, scRNAseq showed upregulation of PU.1 in mice leukemia, suggesting PU.1 as a potential therapeutic target. To assess whether targeted therapy could successfully treat mutIL7R/TLX3-mouse leukemia, we treated mice that had been injected with the thymocytes transduced with mutIL-7R+TLX3 genes. We found that PU.1 inhibition reduced spleen weights, indicating decreasing of leukemia engraftment. Taken together, we generated a high-resolution dataset with detailed information about the transcriptional landscape and immunophenotype of leukemia generated by mutIL-7R+TLX3, a combination that is observed in patient samples, and suggests new therapeutic drug strategies.
Citation Format: Gisele Rodrigues. Cooperation between mutant IL7R and TLX3 generates mixed myeloid lymphoid leukemia from thymocyte progenitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3924.