Myeloid leukemia vulnerabilities at CTCF-enriched
long noncoding RNA loci
Michelle Ng
Martin Luther University Halle-Wittenberg
Lonneke Verboon
Martin Luther University Halle-Wittenberg
Hasan Issa
Martin Luther University Halle-Wittenberg
Raj Bhayadia
Martin Luther University Halle-Wittenberg
Oriol Alejo-Valle
Martin Luther University Halle-Wittenberg https://orcid.org/0000-0002-0988-7416
Dorit Borchert
Hannover Medical School
Konstantin Schuschel
Martin Luther University Halle-Wittenberg
Eniko Regenyi
Free University of Berlin
Dirk Reinhardt
University Hospital Essen
Marie-Laure Yaspo
Max Planck Institute for Molecular Genetics
Dirk Heckl
Martin Luther University Halle-Wittenberg
Jan-Henning Klusmann ( jan-henning.klusmann@kgu.de )
Goethe University Frankfurt https://orcid.org/0000-0002-1070-0727
Genetics Article
Keywords: long noncoding RNA, C-LNC, MYNRL15
Posted Date: August 9th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-727909/v1
1
Myeloid leukemia vulnerabilities at CTCF-enriched long noncoding RNA loci
Abstract word count: 165
Main text word count: 2656
References: 58
Running title: MYNRL15 in myeloid leukemia
Keywords: long noncoding RNA, myeloid leukemia, CRISPR, chromatin architecture
Abstract 1
The noncoding genome presents a largely untapped source of biological insights, including 2
thousands of long noncoding RNA (lncRNA) loci. While some produce bona fide lncRNAs, 3
others exert transcript-independent cis-regulatory effects, and the lack of predictive features 4
renders mechanistic dissection challenging. Here, we describe CTCF-enriched lncRNA loci 5
(C-LNC) as a subclass of functional genetic elements exemplified by MYNRL15, a pan-mye-6
loid leukemia dependency identified by an lncRNA-based CRISPRi screen. MYNRL15 per-7
turbation selectively impairs acute myeloid leukemia (AML) cells over hematopoietic stem / 8
progenitor cells in vitro, and depletes AML xenografts in vivo. Mechanistically, we show that 9
crucial DNA elements in the locus mediate its phenotype, triggering chromatin reorganization 10
and downregulation of cancer dependency genes upon perturbation. Elevated CTCF density 11
distinguishes MYNRL15 and 531 other lncRNA loci in K562 cells, of which 43-54% associate 12
with clinical aspects of AML and 18.4% are functionally required for leukemia maintenance. 13
Curated C-LNC catalogs in other cell types will help refine the search for noncoding onco-14
genic vulnerabilities in AML and other malignancies.
15
2
Main 16
It becomes increasingly clear that the 98% of the human genome that does not encode pro-17
tein nonetheless contains a wide range of functional elements that are vital for cellular home-18
ostasis
1,2
. These include cis-regulatory elements such as enhancers and promoters, insula-19
tors and other determinants of genome topology, as well as a large number and variety of 20
non-protein-coding transcripts. Long noncoding RNAs (lncRNAs) in particular comprise a 21
substantial portion of the noncoding transcriptome
3-5
and in recent years, have emerged as 22
important players in diverse cellular processes and contexts
6-8
. The hematopoietic system is 23
no exception, where lncRNAs have been described to regulate cell programming and fate
9
, 24
and where their dysregulation has been tied to malignancy
10-16
. LncRNAs present a signifi-25
cant opportunity to extend our understanding of human health and disease; however, the 26
fact remains that the vast majority of lncRNA loci lack functional characterization, and may 27
regulate cellular behaviour in ways yet unknown. Indeed, characterization is often a difficult 28
process complicated by cis-regulatory mechanisms unrelated to the transcriptional product
17-
29
23
. Improved functional classification systems are imperative for expediting investigations into 30
lncRNA determinants of pathophysiology, including the search for noncoding oncogenic vul-31
nerabilities. 32
CRISPRi screens of HSPC/AML lncRNAs identify MYNRL15 as a leukemia dependency 33
Aiming to identify lncRNAs that contribute to myeloid malignancy, we began by analyzing a 34
noncoding RNA expression atlas of the human blood system encompassing hematopoietic 35
stem cells (HSCs) and their differentiated progeny, as well as pediatric acute myeloid leuke-36
mia (AML) samples
16
. In addition to stem cell signatures reminiscent of those previously es-37
tablished for protein-coding genes
24-27
, we discovered progenitor- and AML subtype-associ-38
ated lncRNA profiles that could potentially serve as leukemia-specific targets, given their ab-39
sence in HSCs (Fig. 1a). To probe this resource for functionality and find novel AML vulnera-40
bilities, we conducted a CRISPRi-based dropout screen of 480 lncRNA genes from 8 distinct 41
signatures in 6 human leukemia cell lines (Fig. 1b). Five were selected to represent relevant 42
3
cytogenetic subgroups of AML (ML-2, NOMO-1 [KMT2A-rearreanged], SKNO-1, KASUMI-1 43
[standard risk with t(8:21)], M-07E [high risk with inv(16)(p13.3q24.3)]), and we also included 44
the well-studied erythroleukemia line K562. One candidate emerged as crucial in all six cell 45
lines – AC068831.3 (ID: ENSG00000224441 in Ensembl v91 [release 12/2017]), which we 46
renamed MYNRL15 (myeloid leukemia noncoding regulatory locus on chromosome 15; Fig.
47
1c-d, Extended Data Fig. 1). 48
MYNRL15 is a low-abundance, nuclear-enriched transcript (Extended Data Fig. 2a-b) origi-49
nating from chromosome 15, where it is flanked by two protein-coding genes: UNC45A and 50
HDDC3 (Fig. 1c). Given the local effect of the CRISPRi system on nearby genes (Extended 51
Data Fig. 2c), a range of gain- and loss-of-function approaches were necessary in order to 52
delineate the source of the MYNRL15 knockdown phenotype (Fig. 1e-f, Extended Data Fig. 53
2d-g). While CRISPR mediated excision of MYNRL15 recapitulated the effect produced by 54
CRISPRi, repression of the transcript via RNAi and LNA-gapmeRs had little impact on cell 55
viability (Fig. 1e, Extended Data Fig. 2d-f). Both protein-coding neighbors were also dispen-56
sable, as determined via CRISPR-Cas9 mediated knockout of UNC45A and HDDC3, and 57
CRISPRi mediated knockdown of HDDC3 (Fig. 1e, Extended Data Fig. 2d-g). In addition, 58
overexpression of MYNRL15 cDNAs failed to rescue the CRISPRi knockdown phenotype 59
(Fig. 1f). Altogether, these results indicate that neither of the flanking protein-coding genes, 60
nor the MYNRL15 transcript, is responsible for the function of this locus in myeloid leukemia 61
cells, and rather suggest MYNRL15 as an expressed noncoding regulatory locus. 62
Functional dissection of the MYNRL15 locus reveals crucial regulatory regions 63
Given the apparent dispensability of UNC45A, HDDC3, and the MYNRL15 transcript itself in 64
leukemia cells, we hypothesized that MYNRL15 may harbor DNA regulatory elements which 65
drive its leukemia dependency phenotype. To test this hypothesis, we functionally dissected 66
the MYNRL15 locus via complementary CRISPRi and CRISPR-Cas9 screens tiling a 15 kb 67
area centered on MYNRL15. Notably, this area exhibits features characteristic of cis-regula-68
