P487: SMALL NON-CODING RNAS ASSOCIATE WITH CHROMATIN TO MAINTEIN PROPOGATION OF ACUTE MEYLOID LEUKEMIA

Abstract

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the clonal expansion of myeloid progenitors without terminal differentiation. The interplay between genome and epigenome alterations drives aberrant gene network linking to leukemia transformation. Altered transcription may occur at three-dimensional chromatin level and be orchestrated by a combination of multiple regulatory factors. Although some RNA molecules, especially non-coding RNAs, were reported to modulate transcription in leukemia cells, the regulatory role of genome-wide RNAs on transcription through chromatin association remains largely unexplored. Aims: To identify novel RNA molecules as chromatin-association factors critical for leukemogenesis through modulating transcription Methods: We performed in situ Mapping of RNA-Genome Interactome (iMARGI) in a human acute leukemia cell line – MV4-11 cells. Chromatin association of candidate RNAs was verified using Chromatin Isolation by RNA Purification (ChIRP) by means of biotinylated oligonucleotides for target capture. A series of ChIP-seq assays were performed in multiple AML cell lines to profile chromatin occupancy by Fibrillarin (FBL), a catalytic component of C/D box ribonucleoproteins (snoRNPs), as well as to observe the remodelling of chromatin states marked by H3K27ac and H3K9me3 upon shRNA-mediated knockdown of FBL. Loss-of-function experiments using antisense oligonucleotides were performed on candidate chromatin-associated RNAs in AML cell lines, AML primary samples, and CD34+ healthy bone marrow cells. Results: In total, about 52.1 million RNA-chromatin interactions were identified out of deep sequencing of four iMARGI libraries. Whereas the vast majority (50.5 million, 97%) of RNA-chromatin interactome were intra-chromosome, a small fraction (1.6 million, 3%) occurred inter chromosome. Though protein-coding mRNAs predominate the chromatin-associated RNA species, other non-coding RNAs were also captured. Intriguingly, different from protein-coding mRNAs and long intergenic non-coding RNAs (lincRNAs) which mainly form intra-chromosome interactions, two small non-coding RNA classes, small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs), were found with prevalent frequency (62% and 92%, respectively) of inter-chromosome associations. SNORD3A (U3) and SNORD118 (U8) were C/D box snoRNAs ranked top by the number of their associated interactions in MV4-11 cells as well as in other cell types. Chromatin association of U3 and U8 was verified using ChIRP, with the observation of unique chromatin binding for both U3 and U8, and of specific binding motifs of transcription factors at their binding sites. ChIP-seq revealed that the snoRNP FBL occupied only a small set of genes (n=118), and shRNA-mediated knockdown of FBL had a minimal impact on H3K27ac and H3K9me3, suggesting that snoRNA-chromatin interactions may function independently on FBL-associated snoRNPs. ASO-mediated loss of U3 or U8 significantly impairs leukemia cells proliferation and colony forming capacity in multiple AML cell lines and primary AML blast samples, whereas only mildly affects CD34+ healthy cells. Summary/Conclusion: We identified chromatin-associated snoRNAs U3 and U8 which were essential for leukemia maintenance and may serve as therapeutic targets for leukemia eradication.