METTL16 promotes liver cancer stem cell self-renewal via controlling ribosome biogenesis and mRNA translation.

BACKGROUND: While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N6-methyladenosine (m6A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis. METHODS: Liver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling. RESULTS: METTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a (eIF3a) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s). CONCLUSIONS: Our findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency.

RNA m6A reader YTHDF2 facilitates precursor miR-126 maturation to promote acute myeloid leukemia progression.

As the most common internal modification of mRNA, N6-methyladenosine (m6A) and its regulators modulate gene expression and play critical roles in various biological and pathological processes including tumorigenesis. It was reported previously that m6A methyltransferase (writer), methyltransferase-like 3 (METTL3) adds m6A in primary microRNAs (pri-miRNAs) and facilitates its processing into precursor miRNAs (pre-miRNAs). However, it is unknown whether m6A modification also plays a role in the maturation process of pre-miRNAs and (if so) whether such a function contributes to tumorigenesis. Here, we found that YTHDF2 is aberrantly overexpressed in acute myeloid leukemia (AML) patients, especially in relapsed patients, and plays an oncogenic role in AML. Moreover, YTHDF2 promotes expression of miR-126-3p (also known as miR-126, as it is the main product of precursor miR-126 (pre-miR-126)), a miRNA that was reported as an oncomiRNA in AML, through facilitating the processing of pre-miR-126 into mature miR-126. Mechanistically, YTHDF2 recognizes m6A modification in pre-miR-126 and recruits AGO2, a regulator of pre-miRNA processing, to promote the maturation of pre-miR-126. YTHDF2 positively and negatively correlates with miR-126 and miR-126's downstream target genes, respectively, in AML patients, and forced expression of miR-126 could largely rescue YTHDF2/Ythdf2 depletion-mediated suppression on AML cell growth/proliferation and leukemogenesis, indicating that miR-126 is a functionally important target of YTHDF2 in AML. Overall, our studies not only reveal a previously unappreciated YTHDF2/miR-126 axis in AML and highlight the therapeutic potential of targeting this axis for AML treatment, but also suggest that m6A plays a role in pre-miRNA processing that contributes to tumorigenesis.

RNA Modifications in Cancer Metabolism and Tumor Microenvironment.

RNA modifications have recently been recognized as essential posttranscriptional regulators of gene expression in eukaryotes. Investigations over the past decade have revealed that RNA chemical modifications have profound effects on tumor initiation, progression, refractory, and recurrence. Tumor cells are notorious for their robust plasticity in response to the stressful microenvironment and undergo metabolic adaptations to sustain rapid cell proliferation, which is termed as metabolic reprogramming. Meanwhile, cancer-associated metabolic reprogramming leads to substantial alterations of intracellular and extracellular metabolites, which further reshapes the tumor microenvironment (TME). Moreover, cancer cells compete with tumor-infiltrating immune cells for the limited nutrients to maintain their proliferation and function in the TME. In this chapter, we review recent interesting findings on the engagement of epitranscriptomic pathways, especially the ones associated with N6-methyladenosine (m6A), in the regulation of cancer metabolism and the surrounding microenvironment. We also discuss the promising therapeutic approaches targeting RNA modifications for anti-tumor therapy.

TET2-mediated mRNA demethylation regulates leukemia stem cell homing and self-renewal.

TET2 is recurrently mutated in acute myeloid leukemia (AML) and its deficiency promotes leukemogenesis (driven by aggressive oncogenic mutations) and enhances leukemia stem cell (LSC) self-renewal. However, the underlying cellular/molecular mechanisms have yet to be fully understood. Here, we show that Tet2 deficiency significantly facilitates leukemogenesis in various AML models (mediated by aggressive or less aggressive mutations) through promoting homing of LSCs into bone marrow (BM) niche to increase their self-renewal/proliferation. TET2 deficiency in AML blast cells increases expression of Tetraspanin 13 (TSPAN13) and thereby activates the CXCR4/CXCL12 signaling, leading to increased homing/migration of LSCs into BM niche. Mechanistically, TET2 deficiency results in the accumulation of methyl-5-cytosine (m5C) modification in TSPAN13 mRNA; YBX1 specifically recognizes the m5C modification and increases the stability and expression of TSPAN13 transcripts. Collectively, our studies reveal the functional importance of TET2 in leukemogenesis, leukemic blast cell migration/homing, and LSC self-renewal as an mRNA m5C demethylase.

Targeting YTHDF2/MDSCs to improve radiotherapy.

Immunosuppression contributes to tumor-radiotherapy failure, but the mechanism remains elusive. Wang et al.1 reported that ionizing radiation (IR) induces YTHDF2 expression in myeloid-derived suppressor cells (MDSCs) via an IR-YTHDF2-NF-κB circuit, which contributes to MDSC expansion/migration and treatment failure. Genetic depletion or pharmacological inhibition of YTHDF2 overcomes immunosuppression and improves radiotherapy.

QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

The Development of Naringin for Use against Bone and Cartilage Disorders.

Bone and cartilage disorders are the leading causes of musculoskeletal disability. There is no absolute cure for all bone and cartilage disorders. The exploration of natural compounds for the potential therapeutic use against bone and cartilage disorders is proving promising. Among these natural chemicals, naringin, a flavanone glycoside, is a potential candidate due to its multifaceted pharmacological activities in bone and cartilage tissues. Emerging studies indicate that naringin may promote osteogenic differentiation, inhibit osteoclast formation, and exhibit protective effects against osteoporosis in vivo and in vitro. Many signaling pathways, such as BMP-2, Wnt/ß-catenin, and VEGF/VEGFR, participate in the biological actions of naringin in mediating the pathological development of osteoporosis. In addition, the anti-inflammatory, anti-oxidative stress, and anti-apoptosis abilities of naringin also demonstrate its beneficial effects against bone and cartilage disorders, including intervertebral disc degeneration, osteoarthritis, rheumatoid arthritis, bone and cartilage tumors, and tibial dyschondroplasia. Naringin exhibits protective effects against bone and cartilage disorders. However, more efforts are still needed due to, at least in part, the uncertainty of drug targets. Further biological and pharmacological evaluations of naringin and its applications in bone tissue engineering, particularly its therapeutic effects against osteoporosis, might result in developing potential drug candidates.

The roles and implications of RNA m6A modification in cancer.

N6-Methyladenosine (m6A), the most prevalent internal modification in eukaryotic mRNA, has been extensively and increasingly studied over the past decade. Dysregulation of RNA m6A modification and its associated machinery, including writers, erasers and readers, is frequently observed in various cancer types, and the dysregulation profiles might serve as diagnostic, prognostic and/or predictive biomarkers. Dysregulated m6A modifiers have been shown to function as oncoproteins or tumour suppressors with essential roles in cancer initiation, progression, metastasis, metabolism, therapy resistance and immune evasion as well as in cancer stem cell self-renewal and the tumour microenvironment, highlighting the therapeutic potential of targeting the dysregulated m6A machinery for cancer treatment. In this Review, we discuss the mechanisms by which m6A modifiers determine the fate of target RNAs and thereby influence protein expression, molecular pathways and cell phenotypes. We also describe the state-of-the-art methodologies for mapping global m6A epitranscriptomes in cancer. We further summarize discoveries regarding the dysregulation of m6A modifiers and modifications in cancer, their pathological roles, and the underlying molecular mechanisms. Finally, we discuss m6A-related prognostic and predictive molecular biomarkers in cancer as well as the development of small-molecule inhibitors targeting oncogenic m6A modifiers and their activity in preclinical models.

Phosphorylation stabilized TET1 acts as an oncoprotein and therapeutic target in B cell acute lymphoblastic leukemia.

Although the overall survival rate of B cell acute lymphoblastic leukemia (B-ALL) in childhood is more than 80%, it is merely 30% in refractory/relapsed and adult patients with B-ALL. This demonstrates a need for improved therapy targeting this subgroup of B-ALL. Here, we show that the ten-eleven translocation 1 (TET1) protein, a dioxygenase involved in DNA demethylation, is overexpressed and plays a crucial oncogenic role independent of its catalytic activity in B-ALL. Consistent with its oncogenic role in B-ALL, overexpression of TET1 alone in normal precursor B cells is sufficient to transform the cells and cause B-ALL in mice within 3 to 4 months. We found that TET1 protein is stabilized and overexpressed because of its phosphorylation mediated by protein kinase C epsilon (PRKCE) and ATM serine/threonine kinase (ATM), which are also overexpressed in B-ALL. Mechanistically, TET1 recruits STAT5B to the promoters of CD72 and JCHAIN and promotes their transcription, which in turn promotes B-ALL development. Destabilization of TET1 protein by treatment with PKC or ATM inhibitors (staurosporine or AZD0156; both tested in clinical trials), or by pharmacological targeting of STAT5B, greatly decreases B-ALL cell viability and inhibits B-ALL progression in vitro and in vivo. The combination of AZD0156 with staurosporine or vincristine exhibits a synergistic effect on inhibition of refractory/relapsed B-ALL cell survival and leukemia progression in PDX models. Collectively, our study reveals an oncogenic role of the phosphorylated TET1 protein in B-ALL independent of its catalytic activity and highlights the therapeutic potential of targeting TET1 signaling for the treatment of refractory/relapsed B-ALL.

METTL16 drives leukemogenesis and leukemia stem cell self-renewal by reprogramming BCAA metabolism.

N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, is involved in many pathological processes. METTL16 is a recently identified m6A methyltransferase. However, its role in leukemia has yet to be investigated. Here, we show that METTL16 is a highly essential gene for the survival of acute myeloid leukemia (AML) cells via CRISPR-Cas9 screening and experimental validation. METTL16 is aberrantly overexpressed in human AML cells, especially in leukemia stem cells (LSCs) and leukemia-initiating cells (LICs). Genetic depletion of METTL16 dramatically suppresses AML initiation/development and maintenance and significantly attenuates LSC/LIC self-renewal, while moderately influencing normal hematopoiesis in mice. Mechanistically, METTL16 exerts its oncogenic role by promoting expression of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) and BCAT2 in an m6A-dependent manner and reprogramming BCAA metabolism in AML. Collectively, our results characterize the METTL16/m6A/BCAT1-2/BCAA axis in leukemogenesis and highlight the essential role of METTL16-mediated m6A epitranscriptome and BCAA metabolism reprograming in leukemogenesis and LSC/LIC maintenance.

The m6A reader IGF2BP2 regulates glutamine metabolism and represents a therapeutic target in acute myeloid leukemia.

N6-Methyladenosine (m6A) modification and its modulators play critical roles and show promise as therapeutic targets in human cancers, including acute myeloid leukemia (AML). IGF2BP2 was recently reported as an m6A binding protein that enhances mRNA stability and translation. However, its function in AML remains largely elusive. Here we report the oncogenic role and the therapeutic targeting of IGF2BP2 in AML. High expression of IGF2BP2 is observed in AML and associates with unfavorable prognosis. IGF2BP2 promotes AML development and self-renewal of leukemia stem/initiation cells by regulating expression of critical targets (e.g., MYC, GPT2, and SLC1A5) in the glutamine metabolism pathways in an m6A-dependent manner. Inhibiting IGF2BP2 with our recently identified small-molecule compound (CWI1-2) shows promising anti-leukemia effects in vitro and in vivo. Collectively, our results reveal a role of IGF2BP2 and m6A modification in amino acid metabolism and highlight the potential of targeting IGF2BP2 as a promising therapeutic strategy in AML.

RNA N6 -methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer.

RNA N6 -methyladenosine (m6A) modification and its regulators fine tune gene expression and contribute to tumorigenesis. This study aims to uncover the essential role and the underlying molecular mechanism(s) of the m6A reader YTHDC1 in promoting triple negative breast cancer (TNBC) metastasis. METHODS: In vitro and in vivo models were employed to determine the pathological function of YTHDC1 in TNBC metastasis. To identify bona fide YTHDC1 target RNAs, we conducted RNA-seq, m6A-seq, and RIP-seq, followed by integrative data analysis and validation assays. RESULTS: By analyzing The Cancer Genome Atlas (TCGA) dataset, we found that elevated expression of YTHDC1 is positively correlated with poor prognosis in breast cancer patients. Using a mammary fat pad mouse model of TNBC, YTHDC1 significantly promoted lung metastasis of TNBC cells. Through multiple transcriptome-wide sequencing and integrative data analysis, we revealed dysregulation of metastasis-related pathways following YTHDC1 depletion and identified SMAD3 as a bona fide YTHDC1 target RNA. Depletion of YTHDC1 caused nuclear retention of SMAD3 mRNA, leading to lower SMAD3 protein levels. Loss of YTHDC1 led to impaired TGF-ß-induced gene expression, leading to inhibition of epithelial-mesenchymal transition (EMT) and suppressed TNBC cell migration and invasion. SMAD3 overexpression was able to restore the response to TGF-ß in YTHDC1 depleted TNBC cells. Furthermore, we demonstrated that the oncogenic role of YTHDC1 is mediated through its recognition of m6A as m6A-binding defective mutants of YTHDC1 were unable to rescue the impaired cell migration and invasion of YTHDC1 knockout TNBC cells. CONCLUSIONS: We show that YTHDC1 plays a critical oncogenic role in TNBC metastasis through promoting the nuclear export and expression of SMAD3 to augment the TGF-ß signaling cascade. Overall, our study demonstrates that YTHDC1 is vital for TNBC progression by enhancing TNBC cell survival and TGF-ß-mediated EMT via SMAD3 to enable the formation of distant metastasis and highlights the therapeutic potential of targeting the YTHDC1/m6A/SMAD3 axis for TNBC treatment.

RNA-binding proteins in regulating mRNA stability and translation: roles and mechanisms in cancer.

RNA-binding proteins (RBPs) are key players in cellular physiology through posttranscriptional regulation of the expression of target RNA transcripts. By modulating the processing, stability and translation of cancer-related messenger RNA (mRNA) transcripts, a large set of RBPs play essential roles in various types of cancers. Perturbations in RBP activity have been causally associated with cancer development, tumor metabolism, drug resistance, cancer stem cell self-renewal, and tumor immune evasion. Here, we summarize the recent advances in cancer pathological roles and mechanisms of RBPs in regulating mRNA stability and translation with an emphasis on the emerging category of RNA modification-associated RBPs. The functional diversity of RBPs in different types of cancers and the therapeutic potential of targeting dysregulated RBPs for cancer treatment are also discussed.

FTO in cancer: functions, molecular mechanisms, and therapeutic implications.

N6-methyladenosine (m6A) is the most abundant internal modification in mRNA that affects RNA processing, stability, and translation. Discovered as the first RNA m6A demethylase, the fat mass and obesity-associated protein (FTO) is frequently dysregulated and plays important roles in various types of cancers. Targeting FTO holds promising therapeutic significance via suppressing tumor growth, potentiating immunotherapy, and attenuating drug resistance. Here, we review recent advances in our understanding of the functions and underlying molecular mechanisms of FTO in tumor development, cancer stem cell (CSC) self-renewal, microenvironment regulation, immunity, and metabolism and discuss the therapeutic potential of targeting FTO for cancer treatment.

METTL16 exerts an m6A-independent function to facilitate translation and tumorigenesis.

METTL16 has recently been identified as an RNA methyltransferase responsible for the deposition of N6-methyladenosine (m6A) in a few transcripts. Whether METTL16 methylates a large set of transcripts, similar to METTL3 and METTL14, remains unclear. Here we show that METTL16 exerts both methyltransferase activity-dependent and -independent functions in gene regulation. In the cell nucleus, METTL16 functions as an m6A writer to deposit m6A into hundreds of its specific messenger RNA targets. In the cytosol, METTL16 promotes translation in an m6A-independent manner. More specifically, METTL16 directly interacts with the eukaryotic initiation factors 3a and -b as well as ribosomal RNA through its Mtase domain, thereby facilitating the assembly of the translation-initiation complex and promoting the translation of over 4,000 mRNA transcripts. Moreover, we demonstrate that METTL16 is critical for the tumorigenesis of hepatocellular carcinoma. Collectively, our studies reveal previously unappreciated dual functions of METTL16 as an m6A writer and a translation-initiation facilitator, which together contribute to its essential function in tumorigenesis.

DNA N6-methyldeoxyadenosine in mammals and human disease.

N6-methyladenine (6mA) is the most prevalent DNA modification in prokaryotes. However, its presence and significance in eukaryotes remain elusive. Recently, with methodology advances in detection and sequencing of 6mA in eukaryotes, 6mA is back in the spotlight. Although multiple studies have reported that 6mA is an important epigenetic mark in eukaryotes and plays a regulatory role in DNA transcription, transposon activation, stress response, and other bioprocesses, there are some discrepancies in the current literature. We review the recent advances in 6mA research in eukaryotes, especially in mammals. In particular, we describe the abundance/distribution of 6mA, its potential role in regulating gene expression, identified regulators, and pathological roles in human diseases, especially in cancer. The limitations faced by the field and future perspectives in 6mA research are also discussed.

Serum Exosomal lncRNA DLX6-AS1 Is a Promising Biomarker for Prognosis Prediction of Cervical Cancer.

OBJECTIVE: Deregulation of long noncoding RNAs (lncRNAs) is involved in the initiation and progression of cancer. LncRNA DLX6-AS1 is regarded as an oncogene in many cancer types. However, the clinical role of serum exosomal lncRNA DLX6-AS1 in cervical cancer (CC) is poorly known. This study aimed to analyze the diagnostic and prognostic value of serum exosomal lncRNA DLX6-AS1 in CC. METHODS: A total of 114 patients with CC, 60 patients with CIN (cervical intraepithelial neoplasia), and 110 healthy women were enrolled in this study. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to measure the serum exosomal lncRNA DLX6-AS1 levels in all participants. RESULTS: Serum exosomal lncRNA DLX6-AS1 level was significantly elevated in CC patients compared with CIN patients and normal controls. In addition, high serum exosomal lncRNA DLX6-AS1 expression was positively associated with lymph node metastasis, differentiation, FIGO stage, and shortened survival. Patients with high serum exosomal lncRNA DLX6-AS1 expression were more prone to have a relapse. Furthermore, univariate and multivariate analyses suggested that serum exosomal lncRNA DLX6-AS1 was a potential prognostic indicator for overall survival of CC patients. CONCLUSIONS: These findings demonstrated that serum lncRNA DLX6-AS1 might serve as a promising marker for the diagnosis and prognosis prediction of CC.

R-2-hydroxyglutarate attenuates aerobic glycolysis in leukemia by targeting the FTO/m6A/PFKP/LDHB axis.

R-2-hydroxyglutarate (R-2HG), a metabolite produced by mutant isocitrate dehydrogenases (IDHs), was recently reported to exhibit anti-tumor activity. However, its effect on cancer metabolism remains largely elusive. Here we show that R-2HG effectively attenuates aerobic glycolysis, a hallmark of cancer metabolism, in (R-2HG-sensitive) leukemia cells. Mechanistically, R-2HG abrogates fat-mass- and obesity-associated protein (FTO)/N6-methyladenosine (m6A)/YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated post-transcriptional upregulation of phosphofructokinase platelet (PFKP) and lactate dehydrogenase B (LDHB) (two critical glycolytic genes) expression and thereby suppresses aerobic glycolysis. Knockdown of FTO, PFKP, or LDHB recapitulates R-2HG-induced glycolytic inhibition in (R-2HG-sensitive) leukemia cells, but not in normal CD34+ hematopoietic stem/progenitor cells, and inhibits leukemogenesis in vivo; conversely, their overexpression reverses R-2HG-induced effects. R-2HG also suppresses glycolysis and downregulates FTO/PFKP/LDHB expression in human primary IDH-wild-type acute myeloid leukemia (AML) cells, demonstrating the clinical relevance. Collectively, our study reveals previously unrecognized effects of R-2HG and RNA modification on aerobic glycolysis in leukemia, highlighting the therapeutic potential of targeting cancer epitranscriptomics and metabolism.

Targeting FTO Suppresses Cancer Stem Cell Maintenance and Immune Evasion.

Fat mass and obesity-associated protein (FTO), an RNA N6-methyladenosine (m6A) demethylase, plays oncogenic roles in various cancers, presenting an opportunity for the development of effective targeted therapeutics. Here, we report two potent small-molecule FTO inhibitors that exhibit strong anti-tumor effects in multiple types of cancers. We show that genetic depletion and pharmacological inhibition of FTO dramatically attenuate leukemia stem/initiating cell self-renewal and reprogram immune response by suppressing expression of immune checkpoint genes, especially LILRB4. FTO inhibition sensitizes leukemia cells to T cell cytotoxicity and overcomes hypomethylating agent-induced immune evasion. Our study demonstrates that FTO plays critical roles in cancer stem cell self-renewal and immune evasion and highlights the broad potential of targeting FTO for cancer therapy.