LDB1-dependent enhancer connectivity constrains a metabolic synthetic lethality in T-cell acute lymphoblastic leukemia

LDB1 and LMO2, two proteins frequently overexpressed in T-cell acute lymphoblastic leukemia (T-ALL), form a chromatin architectural complex that promotes chromatin looping between enhancers and/or promoters. Here, we defined LDB1-driven oncogenic enhancer connectivity in T-ALL and examined its impact on therapeutic vulnerabilities. To identify proximal LDB1 targets in T-ALL, we engineered isogenic T-ALL cell lines (LOUCY [ETP-ALL] and KOPT K1 [non-ETP-ALL]) with dTAG degrons at the endogenous LDB1 loci. Treatment with dTAG-V1 ligand for 4 hours reduced LDB1 protein levels and chromatin occupancy by >90%. Acute LDB1 loss disrupted spatial enhancer connectivity at critical leukemic oncogenes in both LOUCY (e.g. HHEX, MYB, MYCN ) and KOPT K1 (e.g. DUSP6, STAT4 ), resulting in their downregulation and subsequent reduction in cell growth. CRISPRa-mediated restoration of select LDB1-dependent oncogenes, such as MYB , in LDB1-depleted cells rescued cell expansion. Nascent transcript profiling further revealed that acute LDB1 loss affected distinct gene sets in LOUCY and KOPT K1, consistent with their distinct identities. Notably, however, several cholesterol biosynthetic genes ( HMGCS1 , MVD , MVK ) were upregulated in both cell types. These changes were not driven by altered expression or subcellular localization of SREBP2-the canonical transcriptional regulator of these genes. Instead, they were caused by altered enhancer-promoter connectivity in the absence of LDB1. Specifically, LDB1 loss disrupted connectivity of nearby enhancers for different genes, thereby liberating them to form de novo contacts with HMGCS1, MVK, and MVD gene promoters to activate them. Hence, by clustering regulatory elements, LDB1 not only enables the expression of genes but also constrains enhancers from making inappropriate contacts. Importantly, the native LDB1-dependent loops are detected in primary samples from patients with T-ALL based on published H3K27ac HiChIP data. Functionally, we found that cholesterol flux and expression of upstream regulators, like SREBP2 , increased with prolonged LDB1 depletion, suggesting a feed-forward regulatory mechanism on cholesterol homeostasis in T-ALL. Using both LDB1 degron and knockout models, we observed that LDB1 loss sensitizes leukemic cells to rosuvastatin and pitavastatin by 2-5-fold. This was recapitulated in several T-ALL cell lines with distinct molecular drivers upon LDB1 knockout. Furthermore, statin sensitization was rescued by spike-in of mevalonate or geranylgeranyl pyrophosphate, confirming that this phenotype is driven by altered cholesterol metabolism. Together, our study illustrates a paradigm by which LDB1 loss enables illegitimate spatial connections of enhancers with cholesterol biosynthetic gene promoters. This, in turn, creates a new metabolic addiction in leukemic cells, which may be targeted with statins.