Hottip Lncrna Reinforces CTCF Defined Chromatin Boundaries and Drives Wnt Target Gene Expression in AML Leukemogenesis

We reported recently that HOXA locus associated lncRNA, HOTTIP, is highly expressed in AML patients carrying MLL rearrangement and NPM1^c+ mutations. The expression of HOTTIP positively correlates with posterior HOXA gene expression and poor patient survival. We further demonstrated that HOTTIP acts as an epigenetic regulator to define oncogenic HOXA topologically associated domain (TAD) and drive HOXA associated leukemic transcription program. However, it remains unclear whether and how HOTTIP lncRNA is involved in remodeling leukemic genome to facilitate AML leukemogenesis. Here, we showed that HOTTIP regulates a fraction of CTCF binding sites (CBSs) in the AML genome by directly interacting with CTCF and its binding motifs. We carried out CTCF ChIP-seq and HOTTIP ChIRP (chromatin isolation by RNA purification)-seq comparing WT and HOTTIP knockout (KO) MOLM13 cells. KO of HOTTIP in MLL-rearranged MOLM13 AML cells specifically impaired CTCF binding sites that were co-occupied by HOTTIP lncRNA, whereas loss of HOTTIP did not affect global CTCF binding. These target genes include posterior HOXA genes and Wnt target genes such as C-MYC, EVI1, AXIN, and TWIST1. Furthermore, we found that HOTTIP interacts with its putative target sites by formation of DNA: RNA hybridization structure triple helix and R-loop in vivo and in vitro. We then carried out DRIP (DNA-RNA immunoprecipitation)-seq and DRIPc(DNA-RNA immunoprecipitation followed by cDNA conversion)-Seq, which utilize a sequence independent but structure-specific S9.6 antibody for DRIP to capture global R-loops, by comparing WT and HOTTIP KO MOLM13 cells. The obtained DRIP-seq and DRIPc-seq data were then incorporated and integrated with the HOTTIP ChIRP-seq and CTCF ChIP-seq data to explore global collaboration between R-loop and HOTTIP associated CTCF binding sites. We found that HOTTIP interacts with CTCF binding motif that defines the TADs and the promoters of the HOTTIP target genes by formation of R-loop or triple helix structure. Loss of HOTTIP disrupted the R-loop formation at promoters and enhancers of the HOTTIP target genes to inhibit their expression. In MLL-rearranged AML genome, in addition to the HOXA locus, CTCF forms leukemic specific TADs that protect aberrant Wnt target genes. Depletion of HOTTIP lncRNA impaired CTCF defined TADs in the Wnt target gene loci and reduced Wnt target gene expression. In contrast, overexpression of Hottip lncRNA (Hottip-Tg) in the mice bone marrow hematopoietic compartment perturbs hematopoietic stem cell (HSC) self-renewal and differentiation leading to AML like disease by reinforcing CTCF defined TADs, enhancing chromatin accessibility within TADs, and upregulating gene transcription in the Wnt target loci. Finally, when we treated HOTTIP expressed primary patient AML cells carrying MLL-rearrangement and their derived PDX mouse model with a canonical Wnt inhibitor, ICG-001, ICG-001 inhibited AML LSC self-renewal in in vitro by LTC-IC assays and in vivo leukemogenesis in the PDX mouse models with an aberrant HOTTIP lncRNA expression, but not in HOTTIP negative/low non-MLL AML samples. Thus, HOTTIP lncRNA and CTCF cooperate to specifically reinforce CTCF defined WNT target locus TADs and drive Wnt target gene expression in the HOTTIP expressed AML.