"Nature Cancer": Chinese scientists find that "cancer king" is sensitive to "synthetic death"!

As one of the most common RNA modifications, the methylation of RNA adenine bases at the nitrogen 6 position (m6A) has been under constant interest since it re-entered the field of vision of researchers in 2011 [1].

As the second RNA m6A methyltransferase discovered [2-3], METTL16 has recently become a research hotspot in life sciences [4-5].

Recently, a research team led by Zhenkun Lou of Mayo Clinic Oncology, Jian Yuan of Tongji University School of Medicine in Shanghai, and Kaixiong Tao of Union Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology published the latest research results on METTL16 function in the journal Nature Cancer [6].

They found that METTL16 was able to inhibit homologous recombinant repair
of DNA by interacting with the homologous recombination repair key enzyme MRE11 and inhibiting its exonuclease activity through its bound RNA in a way that does not depend on its methyltransferase function.
In the case of DNA damage, METTL16 can be phosphorylated by ATM kinases, resulting in its conformational changes and reduced RNA binding capacity, which in turn causes MER11 to no longer be inhibited
by METTL16.

Reflected in disease models, such as pancreatic cancer, they found that overexpression of METTL16 increased the sensitivity of pancreatic cancer cells to the PARP inhibitor olaparib, especially when
used in combination with the chemotherapy drug gemcitabine.

Olaparib has been approved by the US FDA in homologous recombinant-deficient pancreatic cancer [7].

However, only about 10% of pancreatic cancer patients have homologous recombinant defective mutations, and most pancreatic cancer patients are not eligible for this therapy
.
Therefore, the discovery that METTL16 has the function of inhibiting DNA homologous recombinant repair is of great significance
for the use of PARP inhibitors in pancreatic cancer patients without homologous recombinant defective mutations.

Screenshot of the first page of the paper

Next, let's take a look at how Lou Zhenkun, Yuan Jian, and Tao Kaixiong's
team conducted this research.

Inspired by research on the role of RNA m6A modification in DNA repair [8-9], the researchers decided to explore the role
of m6A modifying enzyme (METTL3-METTL14, METTL16) in pancreatic cancer DNA damage.

By analyzing the levels of DNA double-strand breaks (DSB) and METTL3-METTL14, METTL16 expression levels in microarray samples of pancreatic cancer tissue, the researchers found that DSB levels were higher
in samples with high METTL16 expression.
In addition, the results of the DSB repair reporting system (DR-GFP/EJ5-GFP) showed that knocking down METTL16 in HEK293T cells significantly increased the efficiency
of homologous recombination (HDR)-mediated DNA repair.
Considering that the effect of METTL16 on DNA damage repair function has not been reported, the researchers decided to explore
it further.

METTL16 expression levels were positively correlated with DSB levels in pancreatic cancer tissues

The researchers first constructed cell lines that knocked out METTL16 and reexpressed METTL16 after knockout, and evaluated
the levels of DSB generated by radiation.

The results showed that after 8 h of radiation treatment cells, the staining level of γ-H2AX (DSB marker) in METTL16-deficient (KO) cells was significantly reduced
compared to wild-type cells.
At the same time, after 4 hours of radiation treatment, the DNA tailing phenomenon of KO was significantly improved
compared with that of wild type.
After reexpressing METTL16, the changes in γ-H2AX levels and DNA tailing were reversed
.

These results suggest that METTL16 has an inhibitory effect
on DNA repair.

Knocking out METTL16 can enhance DNA repair and reduce DSB levels

Protein sequence analysis showed that METTL16 has two potential ATM/ATR (DNA repair associated kinase) phosphorylation sites (Ser419 and Ser455).

Therefore, the researchers speculate that METTL16 may be regulated
by ATM/ATR.
And it turned out to be
.
By analyzing the phosphorylation state of these two sites and constructing a site mutation model, the researchers found that ATM can phosphorylate the Ser419 site on METTL16, which will reduce its inhibitory effect on HDR
.

ATM can weaken its inhibitory effect on HR repair by phosphorylating METTL16

HDR repair of DNA double-strand breaks has three main steps, end resection, 3'-single-stranded invasion homologous template chain, and resolution of Holliday junction
.
So, which step of HDR does METTL16 affect?

The researchers found that after 2 hours of radiation treatment, compared with normally expressed METTL16, METTL16 knockout cells had significantly increased levels of proteins RPA32 and RAD51, and reexpression of METTL16 could reverse these changes
.

The protein RPA32 is a single-stranded DNA-binding protein that binds to 3' single-stranded proteins produced during DNA replication or in the event of DNA damage, thereby acting as a stabilizing single-strand, while an increase in RPA32 staining levels means an increase
in 3'-single-stranded strands in cells.
This suggests that METTL16 inhibits end-of-strand excision
.

In addition, the results of co-immunoprecipitation showed that METTL16 interacted with MRE11 in the key complex MRN-CtIP, a key complex of chain end resection, and radiation treatment could reduce this interaction
.

These results suggest that METTL16 is able to inhibit strand-end excision
during HDR repair DNA by interacting with MRE11.

METTL16 inhibits the chain end excision step in HDR by interacting with MRE11

The question then becomes, how does METTL16 interact with MRE11?

Starting with the two DNA-binding domains present on MRE11 and the four RNA-binding motifs present in METTL16, the researchers demonstrated through DNase/RNase treatment and a series of truncated mutation experiments that METTL16 interacts with MRE11 through RNA bound to these two
.
In addition, the results of extracellular nuclease experiments showed that METTL16-RNA complex inhibited MRE11 nuclease activity
after binding to MRE11.

METTL16 interacts with MRE11 via RNA and inhibits the latter's nuclease activity

As mentioned at the beginning, the main function of METTL16 is to modify
RNA with m6A.
However, the researchers found that compared with wild-type METTL16, METTL16 mutants (F187G), which lost methyltransferase activity, could still interact with MRE11 and inhibit chain end resection, thereby inhibiting HDR efficiency
.
That is, the interaction between METTL16 and MRE11 does not depend on the methyltransferase activity
of METTL16.

In addition, the presence or absence of m6A modifications in the RNA that mediates the interaction between METTL16 and MRE11 has no effect
on this interaction.
On the contrary, the phosphorylation of METTL16 by ATM will reduce its ability to bind to RNA, thereby reducing the interaction
between METTL16 and MRE11.

METTL16's binding to RNA and interaction with MRE11 do not depend on METTL16's methyltransferase activity or RNA's m6A modification

So far, the researchers have determined the role of METTL16 in HDR: after DNA damage activates ATM, ATM phosphorylates METTL16 and causes its conformational change, so that RNA is separated from its original bound state on METTL16, which in turn leads to reduced interaction between METTL16 and MRE11, which is released from the complex and participates in HDR to repair DNA damage
.

Considering the inhibitory effect of METTL16 on HDR and the synthetical lethality of PARP inhibitors on HDR-deficient tumors, the researchers analyzed the killing effect of PARP inhibitor olaparib on BRCA1/2 wild-type pancreatic cancer cells expressing different levels of METTL16
.

The results showed that cell lines with high METTL16 expression were more sensitive to olaparib, knocking out METTL16 in the background of high METTL16 expression reduced the sensitivity of cells to olaparib, and overexpression of METTL16 in the background of low expression METTL16 increased the sensitivity
of cells to olaparib.
This positive correlation between METTL16 expression levels and sensitivity to olaparib has also been demonstrated
in mouse pancreatic cancer models.

METTL16 expression levels in pancreatic cancer cells and tumors were positively correlated with sensitivity to olaparib

Finally, the researchers evaluated the effect
of METTL16 expression levels on the survival of pancreatic cancer patients and on the effectiveness of clinical therapy for pancreatic cancer.

The results showed that the expression level of METTL16 in pancreatic cancer patients was positively correlated
with their survival.
In addition, animal model results showed that tumors with high METTL16 expression were more sensitive to gemcitabine monotherapy and olaparib monotherapy, and tumor development was slowed down
.
In tumors with high METTL16 expression, the combination of gemcitabine + olaparib can cause tumor recession and significantly prolong the survival of
mice.

Pancreatic cancer patients with high METTL16 expression have better survival, which also indicates higher sensitivity to pancreatic cancer treatment drugs

Overall, this study identified a novel function of METTL16, namely that METTL16 inhibits homologous recombination repair
of DNA by interacting with MRE11, a key enzyme for DNA homologous recombination.

At the same time, the study revealed that the level of METTL16 is a potential prognostic indicator of pancreatic cancer, and the combination of gemcitabine + olaparib may be an effective strategy
for the treatment of pancreatic cancer with high METTL16 expression.

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