LIM domain only-2 (LMO2) is one of the most frequently deregulated oncogenes in sporadic and gene therapy- induced human acute T-cell lymphoblastic leukemia (T-ALL). Lmo2 encodes an 18 kDa protein that binds class II basic helix-loop-helix transcription factors (i.e. bHLH: TAL1 or LYL1) and GATA factors (GATA1-2) and LIM domain binding protein 1 (LDB1) in a large multi-subunit complex at promoters and enhancers of hematopoietic stem and progenitor cells (HSPCs). LDB1 binds to LMO2 via its LIM interaction domain (LID) and its homodimerization links near and distant E box-GATA sites in erythroid progenitors. The nature of the LMO2-associated protein complex in T-ALL and LMO2's transcriptional targets in T-ALL are not well characterized. We addressed these questions through a mouse genetic approach. We generated CD2-Lmo2 transgenic mice and defined two distinct subtypes of highly penetrant T-ALLs: one class of leukemias had familiar Notch1 mutation and Notch1-target gene upregulation and the second class closely modeled Early T- cell Precursor (ETP-) ALL, a highly treatment-resistant subtype. ETP-ALLs are derived from developing T-cell progenitors, express antigens of other lineages, have a transcriptional profile that resembles hematopoietic stem cells (HSCs), and express LMO2, HHEX, MYCN, MEF2C, and LYL1 oncogenes. All these features are recapitulated in CD2-Lmo2 transgenic mice including the ETP-ALL transcriptional signature, which is upregulated along with Lmo2-induced hematopoietic stem cell- (HSC) like features of differentiation arrest, relative quiescence, and enhanced self-renewal. In preliminary data, LDB1 was concordantly expressed and co-purified with LMO2 protein in ETP-ALL cells. Using conditional deletion in thymocytes, where Ldb1 is dispensable for T-cell development, we discovered that Ldb1 was required for Lmo2-induced T-ALL; through mutagenesis studies, we found specific residues within the LID of LDB1 that are required for LMO2 binding; most strikingly, LDB1 mutant proteins that could not bind LMO2 were unstable. In fact, LDB1 and LMO2 proteins stabilize each other. Additionally, to address the concordant upregulation of LMO2 with specific oncogenes in ETP-ALL, we performed LDB1 ChIP-seq analysis and discovered occupancy of LMO2 and LDB1 at many genes in the ETP-ALL HSC-like signature including LYL1 and HHEX. We also discovered that Hhex is a required transcriptional target for Lmo2-induced T-ALL. In summary, our preliminary data on Lmo2 and T- ALL establishes an essential protein partner, Ldb1, and an essential target, Hhex, in a highly treatment- resistant leukemia, ETP-ALL. In this proposal, we will test the hypothesis that Ldb1 deletion attenuates T-ALL development by reducing the pre-leukemic target population in CD2-Lmo2 transgenic mice. We will also investigate whether Ldb1 deletion causes destabilization of Lmo2 protein by ubiquitin-mediated proteasomal degradation and disrupts transcriptional regulation of key targets such as Hhex.
The objective of this grant is to understand how the cancer-causing gene, LMO2, functions by associating with partners within leukemia cells. Improved understanding of LMO2's function in leukemia will allow us to design and apply novel therapies for these fatal diseases.
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