HoxBlinc LncRNA Reprograms CTCF Independent TADs to Drive Leukemic Transcription and HSC Dysregulation in NUP98 Fusion Transformed Leukemia

Nucleoporin 98 (NUP98) fusion oncoproteins are implicated in various types of leukemia, particularly high-risk childhood leukemias. These NUP98 fusion-driven leukemias often exhibit unique aberrant HOX gene signatures that play critical roles in transforming hematopoietic stem cells (HSCs). However, the molecular mechanisms underlying NUP98 fusion-driven chromatin landscape and leukemic homeotic transcription remains elusive.

Here we report that a Hoxb-associated lncRNA HoxBlinc were aberrantly activated in NUP98-PHF23 (NP23) fusion-driven primary leukemias. HoxBlinc is known as a key regulator of Hoxb genes, influencing the balance between HSC self-renewal and differentiation. To investigate whether HoxBlinc regulates NP23 epigenetic landscape alteration and signature homeotic oncogenic transcription, we performed chromatin isolation by RNA purification with sequencing (ChIRP-Seq), MLL1/CTCF ChIP-seq, H3K4me3 CUT&RUN, ATAC-seq and HiC-seq in normal B cell precursors and NP23-driven B-ALL cell line. Our results demonstrated that HoxBlinc mainly binds at promoter and occupies 43.3% of upregulated genes in NP23 leukemia, suggesting Hoxblinc can regulate leukemic transcription. Interestingly, HoxBlinc occupied the boundaries of active Hoxa/Hoxb domains where H3K4me3 and ATAC peaks were aberrantly enriched in NP23 leukemia. Moreover, the genes bound by Hoxblinc overlapped with 37.1% of genes that encompassed in upregulated topologically associated domains (TADs). These genes are involved in anterior/posterior pattern specification, hemopoiesis and lymphoid activation. Our data suggested HoxBlinc plays an important role in homeotic/oncogenic TAD formation and leukemic transcription by acting downstream of NP23-driven leukemia.

To explore the regulatory function of HoxBlinc, we utilized CRISPR Cas9 and shRNA approaches to knockout or knockdown HoxBlinc in NP23-related leukemia. Loss of Hoxblinc resulted in a reversion of aberrant homeotic TADs and corresponding chromatin accessibility and gene transcription. Transplantation of NP23-driven leukemia cells deficient of HoxBlinc led to alleviation of NP23-driven leukemogenesis in mouse models. Hoxblinc depletion reduced chromatin accessibility at promoter. Further analysis revealed that promoter regions showing decreases in both HoxBlinc binding and chromatin accessibilities also exhibited impaired recruitment of MLL1 and reduced H3K4me3 peaks. These findings suggest that HoxBlinc is required to maintain leukemic transcription by recruiting MLL1 and reorganizing homeotic/leukemic TADs in NP23-related leukemia. Notably, HoxBlinc depletion reduced its binding at the boundaries of active Hoxa/Hoxb domains, but didn’t affect CTCF binding in these loci. Our data indicates that HoxBlinc reprograms NP23-related oncogenic homeotic TADs in a CTCF independent manner.

Furthermore, transgenic expression of HoxBlinc in the hematopoietic compartment of mice led to the development of AML/BALL disease similar to NP23 transgenic mice. This was accompanied by an increase in B220+/cKit+/Gr1+ population in the bone marrow, and enlarged spleen/lymph node. HoxBlinc transgenic expression induced aberrant homeotic oncogenic TAD topology and transcription. Single cell RNA-seq revealed that overexpression of Hoxblinc in LK cells altered normal hematopoietic differentiation trajectories by specifically enriching myeloid progenitor/pro-B cell populations while blocking erythroid and mature lymphoid cells.

We demonstrated HOXBLINC is overexpressed in primary AML patients harboring NUP98-HOXA9 (NH9) translocation compared with HOXBLINC expressing OCI-AML3 cells. To examine the role of HOXBLINC in NH9-related AML, we performed HOXBLINC knockdown followed by RNA-seq and xenograft studies in NSGS mice. HOXBLINC knockdown led to downregulation of pathways involved in immune system process, multicellular organismal process, and hemopoiesis, consistent with findings in the NP23 transgenic mouse model. NSGS mice receiving HOXBLINC knockdown cells showed significantly prolonged leukemia free survival compared to mice receiving NH9 AML cells. Therefore, our study suggests that HoxBlinc lncRNA is essential for maintaining homeotic oncogenic transcription profile and leukemogenesis driven by NUP98 fusion oncoprotein, by reprograming CTCF independent TAD topology and chromatin signature.