Project Summary/Abstract Despite significant advances in T cell acute lymphoblastic leukemia (T-ALL) therapy, 25% of patients relapse. Recent evidence suggests that failure to eliminate dormant leukemia-initiating cells (L-IC) underlies relapse. Efforts to prospectively isolate L-IC from ALL patients to define the critical pathways of L-IC dormancy and chemoresistance have thus far been unsuccessful. Research from my lab has shown that mouse double negative DN stage 3 (DN3) thymic progenitors are enriched in L-IC that contribute to resistance to Notch targeted therapy, however the L-IC remains an undefined subset of the leukemic DN3 population. To identify the L-IC, we profiled leukemia progression in vivo at the single cell level and uncovered dormant and proliferative DN3 clusters that are transcriptionally distinct. In Aim 1, we will use nucleosome labeling to track the mitotic history of dormant and proliferative DN3 leukemic cells to functionally assess their ability to initiate leukemia and tolerate chemotherapy in vivo. We will also use scRNA-seq to profile chemotherapy response in vivo in order to determine whether chemotherapy enriches for dormant DN3 or selects for additional DN3 heterogeneity. We find Btg2, known to mediate T cell quiescence by deadenylating Myc, enriched in mouse dormant DN3 leukemic cells and in ALL patient label retaining cells and ALL patients with minimal residual disease, leading us to hypothesize that Btg2 regulates dormancy in mouse and human L-IC. We will test this hypothesis in Aim 2 by deleting Btg2 in mouse DN3 cells and examining effects on Myc expression, dormancy and L-IC frequency. Dormancy may also reflect Polycomb repressor complex (PRC) 2 mediated silencing of the Myc enhancer as we find PRC targets enriched in dormant DN3. We will use CUT&Tag to interrogate the Myc enhancer to uncover how Myc is suppressed in these Notch1-active dormant DN3 cells. The work proposed in Aim 3 builds on our siRNA screen to identify glucocorticoid (GC) resistance genes associated with relapse. We identified Estrogen Related Receptor b (ESRRb) as a novel GC resistance gene that we find under-expressed in therapy resistant ALL patients. We demonstrate that ESRRb silencing interferes with GC target gene expression by unclear mechanisms. We will determine if ESRRb increases glucocorticoid receptor (GR) binding and mediates chromatin looping to potentiate GR transcription. Finally, the knowledge gained in mouse T-ALL models will be translated to human T-ALL by determining the effect(s) of ESRRb activation on the GC response and overall survival in GC resistant or relapsed pediatric T-ALL patient-derived xenografts. Together, these studies will advance understanding of T-ALL pathogenesis and therapy failure by identifying the dormant L-IC signature, revealing Btg2 and Myc as critical regulators of L-IC dormancy and by developing novel GC re-sensitizing strategies to prevent relapse in pediatric T-ALL patients.