Defining Oncogenic Transcriptional Networks Regulated By LDB1 in T-Cell Acute Lymphoblastic Leukemia

Despite the discovery of LMO2 translocations in T-cell acute lymphoblastic leukemia (T-ALL) over 30 years ago, the mechanism by which LMO2 contributes to leukemogenesis remains unclear. LMO2 acts as a molecular adaptor bringing the chromatin architectural protein LDB1 into proximity with DNA-binding proteins such as GATA and basic helix-loop-helix transcription factors like TAL1. Our group and others have previously demonstrated that LDB1 is both necessary and sufficient to form long range chromatin loops between enhancers and promoters. Previous studies have found that overexpression of Lmo2 in transgenic mouse thymocytes leads to the development of T-ALL with high penetrance in a manner dependent on LDB1. Thus, we hypothesized that LDB1 aberrantly forms enhancer-promoter loops at oncogenic loci in LMO2-overexpressing forms of T-ALL, thereby driving an oncogenic transcriptional program.

To elucidate the transcriptional network regulated by LDB1, we first profiled LDB1 occupancy in two T-ALL cell lines with high LMO2 expression: LOUCY, an early thymic precursor ALL, and KOPT K1, a near late cortical T-ALL. ChIP-seq demonstrated that the majority of LDB1 peaks fall within enhancer regions. Surprisingly, over 60% LDB1-occupied regions in LOUCY do not overlap with those observed in KOPT K1. Motif enrichment analysis at non-overlapping enhancers in LOUCY also displayed preferential enrichment for HOX and RUNX motifs while those in KOPT K1 were enriched for STAT and NFAT, suggesting that transcriptional miswiring in different T-ALL subtypes may rely on distinct enhancer compositions.

To study the direct effects of LDB1 on gene expression and chromatin folding, we generated isogenic LOUCY and KOPT K1 cell lines harboring homozygous knock-ins of a FKBP12^F36V degron (dTAG), enabling us to degrade LDB1 almost entirely upon dTAG-V1 ligand exposure for 4 hours. Importantly, we did not observe significant changes in chromatin accessibility (ATAC-seq) or enhancer activity (H3K27ac ChIP-seq) upon acute LDB1 degradation, suggesting that architectural and transcriptional changes are attributable to LDB1 loss as opposed to secondary changes due to lost enhancer activity. Furthermore, LMO2 co-occupancy with LDB1 remained relatively unperturbed at these sites further underscoring LDB1 as a direct mediator of chromatin looping.

We performed Micro-C to evaluate chromatin architecture acutely dependent on LDB1. Chromatin compartments and topologically associated domains (TADs) remained intact upon LDB1 degradation. However, we observed widespread changes in chromatin loops, particularly those involving enhancer-promoter and enhancer-enhancer contacts that will be discussed in detail. We next used TT-seq to assess changes in nascent transcription upon LDB1 depletion. Genes regulated by LDB1 in both T-ALL subtypes are the BCL2 family members BCL2L1 and MCL1. The BCL2 family members represent critical anti-apoptotic proteins which are often upregulated in hematologic malignancies and are part of a therapeutic axis sensitive to BH3 mimetic agents. We confirmed that acute degradation of LDB1 leads to a decrease in nascent transcription of these genes, indicating that their expression is in part dependent on LDB1. Despite the commonality of this pathway in both T-ALL subtypes, we also observed distinct patterns of LDB1-mediated transcriptional regulation unique to each cell line associated with developmentally restricted genes. Notably, persistent LDB1 depletion leads to restricted cell growth of LOUCY cells but not KOPT K1 cells, underscoring the differences in the transcriptional programs regulated in these two cell types. Together, these data suggest that LDB1 is a critical transcriptional regulator in LMO2-high T-ALL through regulation of enhancer-promoter looping. Acute perturbation of LDB1-dependent enhancer-promoter connectivity provides novel insights into T-ALL biology and the regulation of genes associated with potentially targetable pathways.

Blobel:Design Therapeutics: Consultancy; Harmonic Discovery: Consultancy; Nucleome Therapeutics: Membership on an entity's Board of Directors or advisory committees.