PU.1 Eviction at Lymphocyte-Specific Chromatin Domains Mediates Glucocorticoid Response in Acute Lymphoblastic Leukemia

Glucocorticoids are an essential part of many multi-agent treatment regimens for lymphoid malignancies where the emergence of glucocorticoid resistance is a significant barrier to a cure. We have previously identified that aberrant chromatin accessibility, at lymphocyte-specific glucocorticoid-response elements (GREs), is associated with drug resistance in acute lymphoblastic leukaemia (ALL). To gain a deeper understanding of regulatory mechanisms leading to these epigenetic alterations, we conducted a multi-omics study, to examine changes in chromatin conformation, following the in-vivo treatment of patient-derived xenografts (PDXs) with glucocorticoid i.e., dexamethasone (DEX). We found that glucocorticoid treatment led to distinct patterns of topological associated domains (TADs) in DEX sensitive and resistant ALLs. Pre-treatment, these TADs were primed by a development-related pioneer transcription factor PU.1, which also occupied GREs in glucocorticoid sensitive but not resistant ALLs. PU.1 binding was associated with lymphocyte-specific activation of GREs and GRE-interacting super-enhancers within the TADs. Furthermore, the PU.1-associated TADs modulated epigenetics and promoted expression of multiple genes, including BIM, ZBTB16, RASA1 and other genes mediating glucocorticoid-induced apoptosis in ALL. The inhibition of PU.1 enhanced sensitivity of ALL cells to glucocorticoid treatment. Overall, this study illustrated a link between the lineage-specific efficiency of glucocorticoids and the developmental epigenetic modulators and provides insights into the role of fundamental epigenetics in clinical practice. Overall design: Hi-C and ChIP-Seq libraries from ALL xenograft pre and post treatment with Dexamethasone. ALL xenograft cells were inoculated by tail-vein injection into NSG mice, and engraftment was monitored weekly as previously described (PMID: 30537513). Mice were randomized and treated with either dexamethasone (15 mg/kg) or vehicle control by intraperitoneal injection when the %huCD45+ cells in the peripheral blood reached 70% for in vitro molecular biology experiments. Briefly, mice were treated when > 70% %huCD45+ cells in the peripheral blood, and sacrificed 8 hours thereafter. Cell suspensions of spleens were prepared, and mononuclear cells enriched to 97% human by density gradient centrifugation. After harvesting, cells were immediately resuspended in fetal calf serum (FCS) containing 10% DMSO, frozen and stored at liquid nitrogen for further use. Hi-C was performed using a modified version of a previously described protocol (PMID: 28435001). Briefly, 10 million cells were collected and cross-linked in 10 ml of PBS containing 1% formaldehyde for 10 minutes at room temperature. The reaction was stopped by 0.125M glycine solution. Cross-linked cell pellet was used to prepare nuclei, which were subjected to digestion with restriction enzyme followed by end-filling to create biotin-labeled blunt ends. Blunt ends were ligated by T4 DNA ligase at room temperature for 4 hours with rotation. Nuclei were digested with proteinase K and 10% sodium dodecyl sulfate (SDS) at 55°C for 30 minutes. Following sodium acetate and pure ethanol precipitation, DNA was isolated and dissolved in Tris buffer. DNA was sonicated using Bioruptor sonicator (Diagenode SA, Belgium), to obtain fragments ranging in size from 150 bp to 300 bp. Sonicated material was then size selected using AMPure XP beads with the 0.55X and 0.3X for HiC. HiC material was then biotinylated, washed, and purified using Dynabeads MyOne Streptavidin T1 beads (Life technologies, 65602). While on beads, DNA ends were blunt ended and dA tailed, followed by ligation of the Illumina sequencing adapter with the T4 DNA ligase. PCR reactions were performed on beads involving 8 cycles of amplification (95°C for 2 min, followed by 94°C for 80 s, 65°C for 30 s, 72°C for 30 s). DNA library was size selected using 1.5% agarose gel to obtain fragments ranging in size from 400bp to 700bp. Sequencing was performed on the Illumina HiSeq 2500 to obtain 150bp paired-end reads. Samples were sequenced to depth of 100 million reads. ChIP-seq were carried out as previously described (PMID: 30537513). Briefly, frozen PDX cells that were harvested from mice after dexamethasone or vehicle control treatment in vivo were revived from cryostorage, and fixed with 1% formaldehyde for 10 min at room temperature. Nuclei were extracted from fixed cells by 10 min incubation in lysis buffer (0.2% NP40 in 10 mM Tris buffer, pH 8.0) followed by centrifugation at 1250 × g for 5 min at 4 °C. Chromatin was fragmented using a Bioruptor sonicator (Diagenode SA, Belgium) on high power at 4 °C with 30 s on/off for 10 min. Separate immunoprecipitates were produced using immunoglobulin raised against the GR (Cell Signaling, Danvers, MA, USA), CTCF (Cell Signaling), Histone (N-terminal acetylated histone H3 in general, Millipore, Billerica, MA, USA; H3K4Me3, H3K27Ac and H3K27Me3, Cell Signaling) and processed according to the manufacturer's instructions. DNA from protein-associated complexes and corresponding input samples was recovered using phenol/chloroform/isoamyl alcohol with phase lock gel tubes (5 Prime, Hilden, Germany). The ChIP DNA samples were dissolved in 20 µl water and sequenced using Hiseq2500 (50SE) platform with 20 M reads per sample.