- N6 Methylation of Adenosine in mRNA by METTL3 Linked to AML
- Linking METTL3 to AML by Gene Knockdown with RNA Interference
- Targeting METTL3 May Lead to a New Drug for AML
Designation of June as Acute Myeloid Leukemia (AML) Month was announced in 2016 by CancerCare®, which provides free emotional and practical support for people with cancer, caregivers, loved ones and the bereaved. The stated goal of the campaign is to put a spotlight on this rare and difficult-to-treat blood cancer that typically affects older adults. In support of that goal, the present blog focuses on newly reported mechanistic findings related to AML.
Taken from Wikipedia.org
By way of an introduction, previous blogs here on RNA epigenetics dealt with discovery of post-transcriptionally modified bases in RNA and enzymes that add (“write”) and remove (“erase”) these modifications, and the effects on RNA function. Among these base-modified RNAs, N6-methyladenosine (m6A; shown here), which is present in bacterial and eukaryotic cells, has been found to have a regulatory role in RNA processing.
According to Wei et al., m6A occurs mainly in the 3' untranslated regions (UTRs) and near the stop codons of mRNA. Dynamic regulation (“editing”) of m6A is found in metabolism, embryogenesis, and developmental processes, indicating an epigenetic regulatory role. For example, it is known that m6A editing is involved in nuclear RNA export, mRNA degradation, protein translation, and RNA splicing. More recently, as summarized below, m6A editing has been linked to human cancer.
m6A Formation in mRNA Mediated by METTL3 Linked to AML
Although m6A is required for differentiation of mouse embryonic stem cells, it has been unknown whether m6A controls differentiation of normal and/or malignant myeloid hematopoietic cells. Compelling evidence in support of such control by m6A was recently published by Vu et al. in Nature Medicine based on collaborative work by a large team of investigators at Memorial Sloan Kettering Cancer Center (NY), Weill-Cornell Medical College (NY), and elsewhere. Readers interested in details should consult this lengthy paper, but for now here’s my short synopsis of what was done.
By way of background, N6-adenosine-methyltransferase 70-kDa subunit (METTL3) is an enzyme that in humans is encoded by the METTL3 gene and, in complex with an analogous subunit (METTL14), is involved in post-transcriptional methylation of the N6 position of adenosine residues in eukaryotic mRNAs to form m6A. The molecular structure and mechanism of how this happens are published and are schematically depicted in the drawing shown below, taken from that work by Śledź and Jinek.
S-adenosyl methionine (SAM); active site loops (ASLs) 1 and 2. Taken from Śledź & Jinek eLife (2016)
Taken from seer.cancer.gov
Since myeloid differentiation (see below) is frequently dysregulated in leukemia, Vu et al. determined if METTL3 expression is altered in leukemia. METTL3 mRNA expression in human AML samples is significantly higher than in other cancer types. To further assess the relative abundance of METTL3 in myeloid leukemia, they examined METTL3 mRNA and protein levels in 11 multiple leukemia cell lines and compared these to primary human cord blood-derived CD34+ cells. METTL3 mRNA was more abundant in AML cell lines (8/11), as was METTL3 protein (11/11). They found no significant difference in METTL3 expression across multiple subtypes of AML.
Taken from dreamtimes.com
After finding increased METTL3 in leukemic cells versus normal cells, they next measured m6A abundance in mRNA and found a significant increase in an AML cell line (MOLM13) compared to CD34+ control cells. These data suggested that elevated m6A might be critical to maintaining an undifferentiated state in myeloid leukemia. To directly address the role of m6A in human myeloid leukemia cells, they examined the effect of METTL3 mRNA knockdown in MOLM13 cells using short hairpin RNA (shRNA). METTL3 knockdown significantly decreased m6A levels, blocked cell growth, induced differentiation and resulted in an increase in apoptosis (aka “programmed cell death”). Similar effects of shRNA-mediated METTL3 depletion were seen in two other AML cell lines.
Vu et al. next investigated whether METTL3 was required in vivo for induction of leukemia. MOLM13 cells were transduced with METTL3-shRNA and transplanted into immunodeficient recipient mice. These cells exhibited delayed leukemia development compared to MOLM13 cells transduced with the scrambled-shRNA. To determine how METTL3 depletion affects m6A -containing transcripts, they performed RNA-Seq on MOLM13 cells following METTL3 knockdown. Consistent with the idea that m6A destabilizes mRNA, transcripts with at least one m6A site showed increased abundance following METTL3 depletion compared to transcripts with no called m6A sites. Moreover, the change in abundance was directly correlated with the number of m6A sites per transcript.
Clinical Relevance
To assess clinical relevance of the MOLM13 m6A profile, Vu et al. mapped m6A in two AML patient samples and compared these transcripts to m6A-enriched transcripts found in MOLM13 cells. They found that the mRNAs with the highest levels of m6A (top 300 transcripts) from the patient samples were enriched with the m6A transcripts from the MOLM13 cells.
Vu et al. stated that this study is the first to demonstrate that m6A is critical for maintaining the differentiation program in the hematopoietic system and that this process is dysregulated in myeloid leukemia. Rather than rephrasing the final conclusion by these researchers, here’s the direct quote:
Our data suggest that inhibition of METTL3 could be exploited as a therapeutic strategy for myeloid malignancies. It is notable that leukemia cells show elevated abundance of METTL3 compared to normal hematopoietic cells. Furthermore, we find that METTL3 depletion shows markedly increased levels of apoptosis in leukemia cells compared to normal cells. These findings suggest a possible therapeutic index. Future studies that target METTL3, potentially in combination with current and emerging therapeutic agents, should be explored.
Parting Comments
Given the increasing interest in elucidation of how mRNAs bearing various types of base-modifications are related to cancer, it seems likely that more discoveries will lead to future therapies of the sort envisaged by Vu et al. for m6A in AML.
Finally, in recognition of June as AML Awareness Month, I thought it would be worth sharing the following information about this disease taken from an authoritative website maintained by the National Cancer Institute.
- The number of new cases of AML was 4.3 per 100,000 people per year, while the number of deaths was 2.8 per 100,000 people per year.
- Lifetime risk of developing AML is ~5% of people will be diagnosed with AML at some point during their lifetime, based on 2013-2015 data.
- Less than 30% of people survive5 years or more after being diagnosed with AML.
- AML is most frequently diagnosed among older people but it effects all age groups.
- Leukemia is cancer that starts in the tissue that forms blood. Most blood cells develop from cells in the bone marrow called stem cells. In a person with leukemia, the bone marrow makes abnormal white blood cells. The abnormal cells are leukemia cells. Unlike normal blood cells, leukemia cells don't die when they should. They may crowd out normal white blood cells, red blood cells, and platelets. This makes it hard for normal blood cells to do their work.
- There are four main types of leukemia: AML, acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML).
Common symptoms for leukemia are as follows:
Taken from pinterest.com
As usual, your comments are welcomed.
