The Epigenetic Regulator Landscape of HOX/MEIS Expression in AML [scRNA-seq]

Aberrant and constitutive activation of the clustered homeobox (HOX) genes and the three-amino-acid loop extension (TALE) domain-containing HOX co-factor MEIS1 (henceforth termed HOX/MEIS) is a recurrent feature in several types of myeloid and lymphoid leukemias. HOX/MEIS misexpression is linked to aberrant self-renewal and therapy resistance in leukemia, but the therapeutic targeting of this important pathway has remained elusive. Using AF10-rearranged leukemia as a prototypical example of HOX/MEIS dysregulation, we sought to comprehensively characterize chromatin regulators that sustain aberrant expression of these genes. We deployed a GFP-MEIS1 knock-in reporter cell line to conduct small-molecule inhibitor screens and a high-density domain-focused CRISPR-Cas9 screen targeting epigenetic regulators. We identified members of at least six distinct chromatin-modifying complexes as HOX/MEIS regulators, including previously characterized HOX/MEIS regulators such as DOT1L, AF10, ENL, and HBO1 as well as less well-characterized and completely novel HOX/MEIS regulators including AFF2, JADE3, casein kinase 2 and the chromatin reader SGF29. These HOX/MEIS regulators were important for the growth of AML cell lines representing diverse leukemia subtypes characterized by HOX/MEIS dysregulation including leukemias with AF10 rearrangements, MLL rearrangements, and NPM1 mutation. Determination of gene expression changes after perturbing each of these MEIS1 regulators in parallel using CROP-seq demonstrated that the deletion of DOT1L, ENL, AFF2, or SGF29 led to the downregulation of several genes associated with stem cell self-renewal and upregulation of differentiation-associated genes. We selected sgRNAs targeting MLLT10, MLLT6, MLLT1, SGF29, MLLT3, CSNK2A1, KAT7, BRD4, DOT1L, CSNK2B, AFF4, KMT2A, AFF2 and a non-targeting control and cloned them into the CROPseq-guide-puro vector (Addgene #86708). Plasmid preparations were mixed in equimolar ratios and the pool was used to prepare lentivirus. U937 cells were transduced in duplicate and sequenced. A total of 18,000 cells per sample were loaded on a Chromium Single Cell Instrument with a targeted capture of 10,000 cells/sample. RNAseq libraries were prepared using the 10X Genomics Chromium Single Cell 3′ v3 Library, Gel Beads & Mutiplex Kit. Sequencing was performed on the Illumina NovaSeq using the S4 200 kit and PE100 run configuration. A custom script was used to deduplicate reads by UMI.