3041 – INVESTIGATING THE ROLES OF LMO2 DURING EARLY T CELL DEVELOPMENT

Abstract

Lmo2 has largely been defined by its oncogenic role in T-cell acute lymphoblastic leukemia; however, Lmo2’s natural role is not fully captured by this overexpression context. We hypothesize that in normal T cell development, Lmo2 contributes to a mechanism that stalls entry into the T cell pathway by initially sequestering E proteins in a TF complex, thus preventing E protein homodimerization which would otherwise push the T cell program forward. To validate, we knocked out Lmo2 in bone marrow-derived progenitor cells and analyzed development in the OP9-Dll1 co-culture system. Previous knockout (KO) experiments were conducted at timepoints where Lmo2 is already downregulated, so we utilized input from the PVA culture system to focus on the earliest developmental stages. Lmo2-KO cells differentiated at least three days faster than controls, measured by cell surface markers, and our initial bulk RNA-seq results confirm this acceleration phenotype: CD25- Lmo2-KO cells upregulate multiple features of the T cell program, including Tcf7, Gata3, Bcl11b, Ets1, Thy1, Rag1, Rag2, Cd3 and significant transcriptional activation of the TCRg and TCRb loci. This could be explained by increased Notch1 and Notch3 expression, thus increasing sensitivity to the Notch ligand-rich environment, though the Notch-response gene Hes1 was not affected. Interestingly, certain progenitor cell program members are Lmo2-activated (Spi1, Mef2c, Bcl11a, Hhex) while other canonical members are not influenced by Lmo2 (Hoxa9, Erg, Flt3), and Lmo2 loss causes specific downregulation of the myeloid signature, including C/EBP family members, Csf3r, Csf1r, Mpo, Elane and Gzma. We explore these findings, along with E protein binding data, to reveal the many roles Lmo2 plays in controlling T cell pathway entry via E protein sequestration, altered Notch signaling and/or the persistence of competing programs.