Pathological reprogramming of the m6A epitranscriptome in uterine fibroids

PROJECT SUMMARY/ABSTRACT
Uterine fibroids (UFs) are the most important benign neoplastic threat to women’s health worldwide. As no long-
term non-invasive treatment option currently exists for UFs, deeper insight into tumor etiology is key to develop
more effective therapies. In this regard, while the epi/genetic determinants of UFs have been characterized
extensively, their underlying pathogenesis nonetheless remains obscure, implicating additional factors in disease
onset. Herein, we propose a novel basis to explain UF development through pathological reprogramming of the
myometrial epitranscriptome and offer proof of concept for therapeutic intervention using inhibitors of tumorigenic
enhancer activity driven by unanticipated m6A-chromatin crosstalk. As the most abundant internal chemical
modification in mRNA, N6-methyladenosine (m6A) is a key determinant of posttranscriptional mRNA fate and
thus cell identity and function. Accordingly, disruption of m6A homeostasis is implicated in a diverse range of
chronic and acute human disease conditions. However, nothing is known about the role of m6A in the
pathogenesis of UFs. We now show that m6A writers METTL3 and RBM15 are aberrantly upregulated in UFs
compared to adjacent myometrium (MM). Further, we show that METTL3 depletion triggers UF cell death and
global upregulation of transcriptionally repressive histone methylation, linking m6A for the first time with UF tumor
biology and revealing its novel crosstalk with the UF epigenome. Integrated RNA methylation and expression
profiling in METTL3-deficient UF cells revealed a profoundly altered m6A modification landscape and identified
high-confidence m6A-modified mRNA effectors of METTL3-driven UF cell growth and survival. Based on these
findings, we hypothesize that aberrant METTL3-dependent RNA methylation reprograms the MM
epitranscriptome, leading to epigenetic dysregulation and altered expression of genes that drive UF initiation and
progression. Accordingly, we propose that epigenetic inhibitors, through suppression of m6A-driven pro-
tumorigenic pathways, will provide therapeutic benefit in UFs. To test these hypotheses we will: (1) Establish the
basis of epitranscriptomic reprogramming in UFs. We will comparatively profile the m6A modification landscape
of mRNAs and chromosome-associated regulatory RNAs (carRNAs) from paired MM and UF tumor tissues and
investigate functional cooperativity between METTL3 and RBM15 in methylomic reprogramming; (2) Delineate
the role of METTL3-dependent RNA methylation in fibrotic transformation. We will ask if METTL3, in a manner
dependent upon its overexpression and methyltransferase activity, can trigger MM stem cell transformation in
vitro and UF tumor formation in vivo; (3) Elucidate the impact of METTL3-dependent RNA methylation on gene
expression in UFs. We will assess the global impact of m6A on mRNA stability and translation as well as carRNA-
dependent control of chromatin state and transcription; (4) Examine the therapeutic potential of BRD inhibitors
in a preclinical mouse model of human UFs. We will evaluate select BRD inhibitors for therapeutic efficacy,
safety, and mechanism of anti-tumor activity, including impact on chromatin status and transcription.

Grant Number: 5R01HD106285-05
NIH Institute/Center: NIH
Principal Investigator: THOMAS BOYER