Expert: METTL3 Is Essential for Growth in Aggressive Chronic Lymphocytic Leukemia

Pharmacy Times® interviewed Lili Wang, MD, PhD, associate professor in the Department of Systems Biology at City of Hope, on her oral presentation at the 114th Annual Meeting of the American Association for Cancer Research (AACR), which looks at how METTL3, an RNA methyltransferase that modifies N6-methyladenosine (m6A) on mRNA and regulates the translation of m6A-installed transcripts, regulates RNA splicing dysregulation and contributes to aggressive chronic lymphocytic leukemia (CLL). The study data presented at AACR showed that METTL3 is essential for CLL growth based on the results of in vitro and in vivo studies.

Pharmacy Times: What is METTL3, and why was it important to focus on in relation to CLL growth?

Lili Wang, MD, PhD: So, METTL3 is an RNA methyltransferase. It works with several proteins to specially deposit adenosine m6A inside of messenger RNA. So, this gene has been shown in multiple cancer types as oncogenes and its upregulation in these cells can usually lead to higher levels of cell proliferation and drive the disease progression. In CLL, we discovered that METTL3 is highly upregulated when compared to normal B cells, and patients with higher level of METTL3 expression usually have poor clinical outcomes, especially when we talk about a particular outcome we’re referring to the shorter time for therapy. So, it looks like this gene is linked to an aggressive type of CLL and may work as an oncogene to drive the disease progression, which of course needs further study.

Pharmacy Times: Why might high METTL3 expression in CLL be associated with poor clinical outcomes?

Wang: This is actually an important question to address. We started to study METTL3, purely from our data mining study. As you may know, my group has a long-term interest in understanding RNA splicing dysregulation in CLL. So, we know that CLL, 20 to 25% of CLL patient samples have RNA spliceosome mutations, and hence, these two are in splicing dysregulation and then resulting in the disease onset. However, in roughly 75 to 80% of CLL samples, they do not have splicing factor mutations, but they have general RNA spicing dysregulation. We have been wondering why. So, we started to explore this question by examining transcriptional and post-transcriptional regulation, by utilizing RNA sequencing, as well as proteomics analysis and from the matched CLL samples. Our results surprisingly discovered that RNA splicing factors are post-transcriptionally upregulated. In other words all the splicing factors are highly expressed at protein level but not at the transcription level, when compared to normal B cells. So, this has been—this discovery is very interesting to us, and it's led us to ask how splicing fractures are upregulated and how these spliceosome proteins can be controlled. So, we first discovered that spliceosome protein abundance is actually an independent risk factor for poor prognosis in CLL through analysis of almost 100, more than 100 CLL patients’ proteomic data. So, these high spliceosome protein expression, these too are RNA splicing dysregulation. So, then we asked what are the key regulators for splicing factor upregulation? Through a correlative study of proteomics data, we found that METTL3 is among the top proteins that have significant correlative protein expression with splicing factors. So, that is when we start to pay attention to METTL3 and try to understand the linkage between METTL3 upregulation, splicing factor upregulation, RNA splicing dysregulation, and CLL.

Pharmacy Times: What did the study results help to elucidate around the role of METTL3 in CLL progression?

Wang: We not only really just focused on, studied the phenotype, but we went one step further. We tried to define the molecular mechanism of how METTL3 may impact splicing factor expression and hence contribute to the disease progression. So, as you may know, that METTL3 has been reported to play a significant role in cancer progression through the translational control, protein translational control. So with these clues, we started with the first step. We performed the experiment, we tried to understand whether or not, if we knock down METTL3 in CLL cell lines, we may see reduction of translation. As we expected, when we knocked down METTL3 in CLL cell lines, we do observe that the translational control is downregulated. In other words, the output of translation is regulated. I think more importantly, we discovered that it is splicing factor transcripts were [sic] affected as compared to other genes. This is number one.

And number two is, when we look at how the METTL3 knock-down may impact the translation, we actually discovered there are specific [sic] dependent regulatory mechanism for which it is well known that 3’-UTR region can be regulated by METTL3. However, we discovered that in the CDS region, we see increased m6A level upon METTL3 knock-down which is kind of surprising, because we are expecting that down regulation of m6A after METTL3 knock-down. So, we went ahead further—tried to analyze the ribosic data, [sic] data, as well as RNA sequencing data from cell line with or without METTL3 knock-down. We were able to pinpoint that the position of m6A at CDS region actually form a roadblock for the translational machinery. Hence, when METTL3 was knocked down, for this specific m6A upregulation in the CDS region, [sic] splicing factor translation. So, they're using 2 different mechanisms to ensure that the splicing factor protein expression is tightly regulated.

Pharmacy Times: What are the potential implications of these study results for CLL treatment development?

Wang: I think as you may know that CLL is a very heterogeneous disease. Patients with CLL, some patients may scramble to the disease within 2 years, while others can live with disease for decades. And then, currently it’s remained incurable. So, identifying aggressive type of CLL will be very, very important. And here of course, we provide another angle to look at aggressive type of CLL. Number one is by looking at METTL3 protein expression level as well as a splicing factor expression level. This would be very important to complement to the current stratification method that’s used the in clinic, for example IGHV mutation status, ZAP70 gene expression level, as well as some somatic mutation patterns. In terms of treatment, so once we are able to identify aggressive type of CLL, the treatment is one of the most important thing that we should consider. Because there are current available METTL3 inhibitor available in the field is ongoing, phase 1 clinical trial is ongoing in AML. So, I hope that through thorough preclinical study as well as some clinical trials, we'll be able to bring the METTL3 inhibitor even into the CLL treatment landscape.

Pharmacy Times: What are future research avenues following these study results?

Wang: Yeah, I think I view this question in 2 ways. One is a biological meaning and the other is the therapeutic treatment. As I just pointed out that METTL3 splicing factor, regular [sic], this is a very novel in CLL, whether or not this is a common regular axes in other type of cancer, especially in the [sic] this needs to be explored. And for which we have some data and we also included in the publication, for the mantle cell lymphoma, we also observed that this regular axi is there [sic] that we need to fully understand if it is there. So, then targeting METTL3 with METTL3 inhibitor would be very important approach to treat the disease as well as to correct RNA splicing dysregulation. So, that's from a second meaning, because the RNA splicing dysregulation, if this is one way to regulate, and there could be other avenues that people can think about to leverage in the METTL3 regulation for RNA splicing regulation. So, that's from the therapeutic treatment. From the biological insights, we have been having a lot of interest trying to understand whether or not a METTL3 is oncogene in CLL, and then whether or not how exactly the METTL3 may contribute to the CLL progression, even though we have already using clinical data and then some of the cell lines demonstrated that it leads to the progression. So, whether or not exactly and through which RNA splicing [sic] that eventually can lead to the disease progression, that part needs to have more further study.