ABSTRACT Recent advances in chromatin conformation capture techniques have revolutionized our understanding of chro- matin organization and have provided novel insights at an unprecedented level of detail. Several studies have identified biologically-relevant structures in DNA-DNA contact maps, such as A/B compartments, topologically- associating domains (TADs), insulated neighborhoods, and have elucidated the role of chromatin architecture in gene regulation and maintenance of cell identity. A handful of very recent studies from our lab and others have shown that aberrant TAD activation or “rewiring” promoter-enhancer interactions can promote cancer growth. However, no study has yet addressed the disruptions of chromatin organization on a genome-wide scale in cancer patients or how such disruptions modify the promoter-enhancer landscape leading to drug resistance and relapse. Using primary acute leukemia patient samples, we have, for the first time, identified recurrent TAD disruptions in leukemia involving key oncogenes (e.g. NOTCH1, MYC) and their targets. For example, we iden- tified a recurrent disruption of 3D chromatin topology in the MYC locus at a previously uncharacterized non- coding CTCF-bound region that insulates MYC from downstream enhancers. This disruption enables chromatin interactions between the MYC oncogene and the downstream enhancers leading to an increase in MYC expres- sion. Based on our preliminary results, we propose to investigate 3D chromosomal landscape reorganization as a new mechanism of cancer initiation, progression and relapse, and to discover novel non-coding regulatory elements (enhancer 3D hubs and their TAD boundaries) that drive leukemia. To this end, we will first profile and analyze a large cohort of leukemia patients using Hi-C and H3K27ac HiChIP both at diagnosis and at relapse to identify recurrent relapse-specific 3D reorganization events. We will combine computational methods with or- thogonal CRISPR strategies to discover transcription factors and epigenetic modifiers that enable the emergence of drug resistance via the rewiring of enhancer-promoter chromatin looping. We will then focus on enhancer hubs: we and others have shown that enhancers that are densely connected with target promoters and other enhancers (i.e. enhancer hubs) are robust regulators of gene expression and their disruption can impact the regulation of multiple genes. Based on these findings, we will test whether such enhancer hubs and their 3D topology can be drivers of drug resistance by activating oncogenic loci in vitro and in vivo. Our proposed study will not only elucidate the role of 3D architecture in leukemia at diagnosis and relapse, but it will also advance our understanding of resistance to therapy and develop new approaches to overcome it.

PROJECT NARRATIVE Despite dramatic improvements in outcome for children with acute lymphoblastic leukemia (ALL), up to 25% of children fail frontline therapy and their prognosis is dismal. While patients with prolonged remission have a better prognosis, most eventually succumb to their disease due to the emergence of resistant clones, despite increased intensity of frontline therapies. In this study, we will investigate how the reorganization of 3D chromatin architec- ture contributes to patients’ relapse in acute pediatric leukemia.