BCR::ABL1-induced enhancer reprogramming uncovers hypersensitivity of Ph+B-ALL cells to enhancer-targeting drugs [ChIP-Seq]

To better understand how BCR::ABL1 establishes the Ph+B-ALL-defining transcriptional program, we adopted an integrative multi-omics approach to study the transcriptome, enhancer activities, and 3-dimensional interactions of enhancers with target genes in Ph+B-ALL cells. We performed an in-depth analysis of cells from human Ph+B-ALL patients and a murine Ph+B-ALL model using ChIP-Seq, RNA-Seq, and Hi-C-based methods to link enhancers to the promoters they regulate. We further interfered with enhancer function to explore their role in Ph+B-ALL and investigated BCR::ABL1-regulated TFs that are recruited to them using targeted proteasomal degradation and RNA interference (RNAi). Overall design: This dataset contains several different experimental designs: (1) Monitoring of enhancer activation during BCR::ABL1-induced malignant transformation using murine primary B-cell precursors from Trp53bp1-/- mice: H3K27ac ChIP-Seq was done on primary B-cell precursors from Trp53bp1-/- mice cultured for 7 or ~60 days and either transduced with BCR::ABL1(p210)-encoding retrovirus or empty vector (EV) as controls. Culture was done in presence of mIL3 throughout. (2) Monitoring of enhancer activation during BCR::ABL1-induced malignant transformation using murine primary B-cell precursors from C57BL/6 mice: H3K27ac ChIP-Seq was done on primary B-cell precursors from C57BL/6 mice cultured for 3 or ~60 days and either transduced with BCR::ABL1(p190)-encoding retrovirus or empty vector (EV) as controls. Culture was done in presence of mIL3 throughout. For successfully transformed BCR::ABL1(p190) expressing cells (at ~D60), cells were additionally treated with 10nM Ponatinib or DMSO as control for analysis of BCR::ABL1 kinase-dependent enhancer activation. (3) Comparison of enhancer activation in human Ph+B-ALL cells vs normal bone marrow B-cell precursors: H3K27ac ChIP-Seq was done on Ph+B-ALL cell lines (SUP-B15, TOM-1, BV-173) or primary leukaemia cells (BAL05, BAL08, BAL11), and bone marrow B-cells purified from healthy mononuclear bone marrow cells (obtained from Cambridge Bioscience). (4) Analysis of the effect of BCR::ABL1 kinase inhibition by Ponatinib on enhancer activation in human Ph+B-ALL cells for comparison to PCHI-C and RNA-Seq data: H3K27ac ChIP-Seq was done on Ph+B-ALL cell lines and primary cells (SUP-B15, TOM-1, BAL08) treated for 24h with DMSO or 100nM Ponatinib. IgG ChIP-Seq and/or Input libraries were generated as controls for peak calling. Primary BAL08 cells were co-cultured on mitotically-inactivated OP9 feeder cells for 3 weeks before experiment. (5) Analysis of the effect of BCR::ABL1 kinase inhibition on enhancer activation in human Ph+B-ALL cells for using Asciminib: H3K27ac ChIP-Seq was done on Ph+B-ALL cell lines (SUP-B15, TOM-1, BV-173) treated for 24h with DMSO or 1uM Asciminib. (6) Analysis of the effect of BCR::ABL1 kinase inhibition by Ponatinib on enhancer activation in human CML cells: H3K27ac ChIP-Seq was done on CML cell lines (K562, KCL-22, LAMA-84) treated for 24h with DMSO or 100nM Ponatinib. (7) Comparison of the effect of the BCR::ABL1-induced TFs MYC, STAT5, ETV5 on enhancer activation vs BCR::ABL1-induced enhancer activation in human Ph+B-ALL cells: H3K27ac ChIP-Seq was done on Ph+B-ALL cell lines (SUP-B15 and BV-173) that were either treated with Ponatinib (24h, 100nM), the STAT5 degrader AK-2292 (24h, 2.5uM), the MYC degrader A80.2HCl/MYC degrader 1 (24h, 25nM), and/or expressing shRNAs against ETV5 or respective C911 negative control shRNAs. Please note: H3K27ac and respective IgG ChIP-Seq were performed on 15 mio or 1 mio cells depending on cell availability but with consistency within each experimental comparison. ChIP-Seq for MYC, STAT5 and respective IgG controls were performed using 50 mio cells throughout. Specifically, ChIP-Seq libraries for GSM9037533-GSM9037555, GSM9447961-GSM9447967, GSM9447971-GSM9447974 and GSM9447978-GSM9447983 were generated using 1 mio cells, GSM8577378-GSM8577427 and GSM9447984-GSM9447989 using 15 mio cells, and GSM9447968-GSM9447970 and GSM9447975-GSM9447977 using 50 mio cells.