Systematic characterization of the HOXA9 downstream targets in MLL-r leukemia by non-coding CRISPR screens [ATAC-Seq II]
Accumulating evidence indicates that HOXA9 dysregulation is sufficient and necessary for leukemic transformation. Leukemia subtypes showing HOXA9 overexpression include those carrying MLL gene rearrangements (MLL-r), NPM1c mutations, and other genetic alterations. HOXA9 protein is a poor therapeutic target as it lacks targetable pocket domains. Therefore, understanding HOXA9's functional downstream genes and its regulation might provide alternative therapeutic targets. However, it remains largely unknown how HOXA9, as a homeobox transcriptional factor, binds to noncoding regulatory sequences and controls the downstream genes in MLL-r leukemia and across leukemias of other genetic subtypes. In this study, we have successfully conducted dropout CRISPR screens in the MLL-r SEM cell line stably expressing Cas9 against ~1,800 HOXA9 binding peaks. Integrative data analysis identified six reproducible noncoding hits, including a positive control located in the distal enhancer of FLT3. Cas9-editing and dCas9-KRAB silencing HOXA9's binding site in distal region of FLT3 reduced transcription and impaired cell proliferation in vitro and in vivo. In addition, RNA-seq and Q-PCR analysis further identified the functional relevant downstream genes upon CRISPR editing of other candidate HOXA9-bound noncoding segments, including survival essential genes as XBP1, JUN and BAHCC1. In summary, the work is significant as it will advance our understanding of how HOXA9-associated transcription programs reconstruct the regulatory network specifying MLL-r dependency. Moreover, our study will promote the future development of HOXA9-mediated noncoding regulation, and alternative therapeutic targets in HOXA9-driven (including HOXA9 and possibly NUP98-HOXA9 subtype) leukemia in patients. Overall design: ATACseq were performed in SEM for different sgRNA target region to check chromatin accessibility