Disregulation of CTCF Boundary at the HOX Locus Enhances Sensitity to Multiple Drug Treatment in Myeloid Malignancies

Dysregulation of HOX genes is a dominant mechanism of leukemic transformation by altering self-renewal and differentiation properties of HS/PCs leading to leukemic transformation. The overexpression of posterior HOXA9 gene is also a poor prognostic marker in leukemia patients. Recent studies revealed that CTCF insulator constrains temporal HOX gene expression patterns within confined chromatin domains suggesting that CTCF mediated chromatin boundary activity may involve in regulating HOX gene patterns during AML prognosis.

To investigate the role of CTCF in aberrant HOX gene expression and AML prognosis, we employed CRISPR-Cas9 genome editing technology to delete 47 base pairs of the core CTCF motif located between HOXA7 and HOXA9 genes (CBS7/9) in different subtype of AML cells with either MLL-rearrangement or NPM1 mutation. RNA-seq analysis revealed that transcription factors, HOXA9 , RUNX1 , and ZEB1 , as well as MAPK signaling pathway, all of them are involved in AML survival, prognosis and chemo-resistance, are significantly dowregulated in response to the CBS7/9 boundary deletion. Given that these genes and pathways are involved in AML prognosis and chemo-resistance, we then examined whether perturbation of CBS7/9 chromatin boundary resulted in alteration of drug resposes of AML cells. Both WT AML cells and the CBS7/9 defected AML cells derived from MLL-AF9 rearranged MOLM-13 and NPM mutated OCI-AML3 cells were treated with 1 mM of Ara-C or 100 nM of bromodomain (BET) inhibitors. Compared to the WT controls, the defected CBS7/9 boundary resulted in a significantly increase in AML sensitivity to both Ara-C and BET inhibitor treatment in both MOLM-13 and OCI-AML3 cells as evidence by significantly reducing percentage of proliferating cells.

To further examine if treatment related drug sensitivity in AML cells is depended on the levels of HOXA9 expression, we overexpressed HOXA9 cDNA in the CBS7/9 deleted MOLM-13 and OCI-AML3 cells. Gain-of-function (GOF) expression of HOXA9 in the CBS7/9 defected AML cells rescued and restored the resistance to the Ara-C treatment of the CBS7/9 KO cells to the levels compatible to that of the WT control MOLM-13 and OCI-AML3 cells, but HOXA9 overexpression did not rescued the resistance of the CBS7/9 KO AML cells to the BET inhibitor treatment. The data suggest that HOXA9 is directly associated with cytotoxic drug Ara-C resistance, but not BET inhibitor mediated drug responses. KO of CBS7/9 specifically decreased enrichment of H3K9ac and H4K5ac, as well as recruitment of BRD4 to the posterior HOXA domain. Furthermore, overexpression of HOXA9 gene in the CBS7/9 KO AML cells indeed rescued the mRNA transcripts affected by CBS7/9 deletion to the levels compatible to that of WT control MOLM-13 or OCI-AML3 cells. Among rescued genes, ZEB1, STAT5A, and RUNX1 play essential roles in pathogenesis of myeloid malignancies and chemo-drug sensitivity.

Finally, in order to further explore biological role of CTCF boundaries in AML pathogenesis, we analyze the whole genome sequencing data of 183 primary AML patients obtained from public available Cosmic Database for somatic mutations occurred in the CTCF binding sites. Several core CTCF motifs in the HOXA locus, including CBS5/6 and CBS7/9 boundaries, are highly mutated in AML patients. Thus, disruption of CTCF boundary located between HOXA7 and HOXA9 genes perturbs HOXA9 oncogene transcription and regulates chemo- or epigenetic drug sensitivity of acute myeloid leukemia. Alteration of CTCF boundaries in the oncogene loci may provide a novel therapeutic target for treatment of myeloid malignancies.