METTL3 drives telomere targeting of TERRA lncRNA through m⁶A-dependent R-loop formation: a therapeutic target for ALT-positive neuroblastoma

Abstract

Telomerase-negative tumors can maintain telomere length by alternative lengthening of telomeres (ALT) but the mechanism behind ALT is poorly understood. Aggressive Neuroblastoma (NB), in particular, relapsed tumors are positive for ALT (ALT+) which suggests that better dissection of the ALT mechanism could provide novel therapeutic opportunities. TERRA long non-coding RNA (lncRNA) which is derived from the telomere ends is localized to telomeres in R-loop dependent manner and is essential for telomere maintenance. In the present study, we provide evidence that RNA modification at the N6 position of internal adenosine (m6A) in TERRA RNA by methyltransferase METTL3 is essential for telomere maintenance in ALT+ cells and that loss of TERRA m6A/METTL3 leads to telomere damage. We observed that R-loop enriched TERRA is abundantly m6A modified and m6A mediated recruitment of hnRNPA2B1 to TERRA RNA is essential for R-loop formation. Our data suggest that m6A drives telomere targeting of TERRA via R-loop and this m6A mediated R-loop formation could be a widespread mechanism utilized by other chromatin-interacting lncRNAs. Furthermore, treating ALT+ NB cells with METTL3 inhibitor leads to compromised telomere targeting of TERRA and accumulation of DNA damage over telomere, suggesting METTL3 inhibition could be a therapeutic opportunity for ALT+ NB.

Recruitment of the m6A/Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48

“…Recently, it was shown that telomeric repeat-containing RNA (TERRA), which is transcribed from telomeres and functions in telomere maintenance through R-loop formation, is m6A-modified in a METTL3-depndent manner (11, 49). Considering that ZBTB48 regulates telomere metabolism (40, 41), we examined whether ZBTB48 modulates TERRA m6A in an FTO-dependent manner.…”

METTL3/MYCN cooperation drives neural crest differentiation and provides therapeutic vulnerability in neuroblastoma

“…Next, we characterized the pattern of m 6 A modifications in hESC and tNCC using m 6 A RIP-seq (19). We observed that consistent with the upregulation of METTL3/14, a higher number of m 6 A peaks was seen in tNCC compared to hESC ( Fig.…”

“…Human embryonic stem cell line WA09 (H9) obtained from Dr. Fredrik H. Sterky (Sahlgrenska University Hospital, Gothenburg, Sweden) were cultured on Matrigel-coated plates with iPS-Brew XF (Miltenyi) media. To differentiate hESC to trunk neural crest cells (tNCC), hESC were dissociated using Accutase and seeded (day 0) on Matrigel-coated plates to induce differentiation to neuromesodermal progenitor cells (NMP, day 3) which were then driven to tNCC (days 7-9), sympathoadrenal progenitors (SAP, day 12), and further towards sympathetic neurons (SN, day [19][20][21][22][23][24][25] as described previously (15,16). For neural crest stem cells (NCSC) induction, hESCs were dissociated using Accutase and seeded (day 0) on Matrigel-coated plates at a density of 2x10 4 cells/cm 2 in iPS-Brew XF media with ROCK inhibitor (Y-27632 10 μM).…”

“…This fragmented RNA was either used for RNA-seq or m 6 A RIP-seq. m 6 A RIP was performed as previously described (5,19,25) with m 6 A antibody. Input and m 6 A RIP RNA were used to generate sequencing libraries using SMARTer Stranded Total RNA-Seq kit V2, Pico Input Mammalian (Takara Bio).…”

“…METTL3 has been shown to have a tumor promoting role in many cancers (17) and its inhibition through small molecules has recently been proposed as a therapeutic strategy for acute myeloid leukemia (AML) (18). A role for METTL3 mediated m 6 A modification was recently reported in Alternative Lengthening of Telomeres-positive (ALT+) NB (19) but its function is unknown in other types of high-risk NB. High expression levels of METTL3 are predictive of an inferior outcome for NB patients and METTL3 is expressed in both ALT+ and high-risk MYCN-amplified NB tumors suggesting it may have broader relevance (19).…”