LKB1 Loss Upregulates ALKBH5 and Contributes to Aggressive Phenotypes of KRAS Mutated Lung Cancer

Background: Oncogenic KRAS mutations combined with loss of the LKB1 tumor-suppressor gene (KL) are strongly associated with aggressive forms of lung cancer. N6-methyladenosine (m6A) in mRNA is a crucial epigenetic modication that controls cancer self-renewal and progression. However, the function, regulation and mechanism of m6A in this aggressive phenotypes remain largely unclear. Methods: The clinic-pathological role of m6A was evaluated in a cohort of lung cancer tissues and further validated by public databases and integrating bioinformatics analyses. We examined the upstream and downstream regulation of ALKBH5 (AlkB family member 5, an m6A demethylase) using quantitative real-time PCR, western blot, bisulte genome sequencing, luciferase reporter assay, methylated DNA immunoprecipitation, m6A-RNA Immunoprecipitation (m6A-RIP) and m6A-seq data analysis. Results: We found that LKB1 loss decreased m6A levels and correlated with disease progression and poor survival for KRAS mutant lung cancer patients. The effect on m6A levels and the disease progression and survival was mediated by increasing levels of ALKBH5. LKB1 inactivation increased ALKBH5 transcription in multiple KRAS mutated tumor types, including colon, pancreas, and lung. Conversely, LKB1 overexpression decreased DNA methylation of the CTCF-binding motif on the ALKBH5 promoter, which enhanced CTCF binding and inhibited histone modications, including H3K4me3, H3K9ac, and H3K27ac. ALKBH5 demethylation of m6A stabilized oncogenic drivers, such as SOX2, SMAD7, and MYC, through a pathway dependent on YTHDF2, an m6A reader protein. Conclusions: Loss of LKB1 in KRAS mutated cancers promoted ALKBH5 transcription, decreased m6A levels, and increased the stability of m6A target oncogenes, thus contributing to aggressive phenotypes of KRAS mutated lung cancer. vectors and SV-40-Renilla-Luc in the presence of Lipofectamine 2000 Reagent (Invitrogen). After a 24-h transfection, we prepared cell extracts with passive lysis buffer. Luminescence was measured with the Dual-Luciferase Reporter Assay System (Promega), according to manufacturer’s instructions. The relative luciferase reporter activities were normalized to that of Renilla. We performed experiments for each vector as biological triplicates with six technical repeats.

N6-Methyladenosine (m6A) mainly occurs at the consensus motif of GG m6 ACC, and is the most prevalent internal chemical modi cation of mRNAs in eukaryotes [13]. Functionally, the reversible m6A modi cation of mRNAs is critical to cancer self-renewal and malignancy of several tumors [14][15][16][17], including glioblastoma [18], acute myeloid leukemia [19], hepatocellular carcinoma [20], and breast cancer [21]. In this regard, investigation of the landscapes and functions of m6A modi cations is an emerging research frontier known as RNA epigenetics or epitranscriptomics. AlkB homolog 5 (ALKBH5), a demethyltransferase of m6A, is more highly expressed in most tissues than other m6A modulators (8).
Alkbh5 de ciency leads to compromised spermatogenesis in mice, and displays widespread mRNA methylation and global RNA instability [22]. Most recently, ALKBH5 was found to have oncogenic roles in glioblastoma and breast cancer cells (17,20), suggesting it contributes to mRNA m6A methylation in cancer.
This study investigated the roles and underlying mechanisms of m6A modulation in aggressive KL mutant lung cancer cells. We found that loss of LKB1 activity promotes ALKBH5 transcription via DNA methylation and stabilization of m6A target oncogenes in KRAS mutant cancers, which may in uence how vulnerable the cancer is to therapy.

Human lung cancer specimens and cell lines
We obtained fresh and para n-embedded lung cancer specimens from 72 patients who underwent lung cancer surgery between January 2016 and September 2019 at the Cancer Hospital of Harbin University Medical College, China. Clinical characteristics of patients were retrospectively analyzed. Two pathologists performed blinded histological con rmation by hematoxylin and eosin (H&E) staining. The clinical pathology variables of lung cancer patients are summarized in Additional table S1. We collected all clinical samples with informed consent according to Health Insurance Portability and Accountability Act (HIPAA)-approved protocols. The use of human patient samples was approved by the Harbin University Medical College Ethics Committee.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
We extracted total cellular and tissue RNA using TRIzol Reagent (Thermo Fisher Scienti c, USA) and used 1 µg total RNA for reverse transcription using the iScript™ cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instructions. We performed qRT-PCR was performed with 2 × SYBR Green qPCR Master Mix (Bimake, USA) with a CFX96 Touch™ Real-Time PCR detection system (Bio-Rad Inc. USA). We calculated the relative gene expression using the comparative CT method and β-actin RNA sequences as a control. Primer sequences are listed in Additional table S2.

Immunohistochemistry (IHC) staining
We performed IHC analysis of para n-embedded lung cancer tissues containing primary tumors and matched normal lung tissues as previously described [24,25]. In brief, we de-para nized and rehydrated human tissue sections to retrieve antigens by microwaving the sections in 10 mM Sodium Citrate (pH 6.0), and then incubating in 1% hydrogen peroxide to suppress endogenous peroxidase activity. After blocking in 2.5% horse serum for 1 h, we applied primary antibodies to the slides at 1:500 (anti-LKB1, METTL3, METTL14, WTAP, and FTO antibodies), 1:200 (anti-ALKBH5 antibody), and 1:1000 (anti-m6A and 5-mC antibody) dilutions and incubated at 4°C overnight. We stained slides with EnVision + Dual Link System-HRP (Dako) for 1 h at room temperature, then washed, counter-stained with hematoxylin, dehydrated, treated with xylene, and mounted the samples. Images of stained cells in four random elds were captured by using an optical microscope (Olympus, Japan). Relative protein expression were evaluated by a Histoscore (H-score) system. This semiquantitative approach was calculated as the product of the percentage of positive cells by the staining intensity (graded as: 0, non-staining; 1, weak; 2, median; or 3, strong using adjacent normal mucosa as the median). Possible scores ranged from 0 to 300. The results were evaluated by two independent pathologists.

Luciferase reporter assays
For the ALKBH5 promoter reporter assay, we generated serial ALKBH5 promoter reporters by PCR ampli cation and inserted into pGl3-basic plasmid, as we previously reported [23]. We deleted the CTCF region using the Q5 Site-Directed Mutagenesis Kit (NEB) per the manufacturer's protocol. Fire y luciferase activity was used to evaluate the effect of m6A modi cation on SOX2, SMAD7, and MYC activation. We used the pmirGLO Dual-Luciferase miRNA target expression vector from Promega to construct the reporter plasmid, which contained both a re y luciferase and a Renilla luciferase. The wild-type SOX2, SMAD7, and MYC reporter plasmid was individually cloned by inserting the fragment containing the m6A peak region after the re y luciferase coding sequence. We constructed mutant three reporter plasmids by replacing the adenosine bases within the m6A consensus sequences with cytosine. All constructs were For DNA methylation analysis, we used BGS method, as previously described [26,27]. Brie y, 500 ng of genomic DNA was bisul te converted using the BisulFlash DNA Modi cation Kit (Epigentek) following the manufacturer's instructions. We ampli ed the fragment containing CTCF peak region of ALKBH5 promoter using primers listed in Additional table S2. PCR products were puri ed and cloned into a pCR4 TOPO vector using the TOPO TA Cloning Kit (Thermo Fisher Scienti c, Rockford, IL, USA). We isolated and sequenced plasmid DNA from 10 randomly selected clones (Genewiz, Piscataway, NJ, USA).

Methylated DNA immunoprecipitation (MeDIP) analysis
To con rm BGS results, we performed MeDIP using the Methylamp Methylated DNA Capture Kit (EpiGentek) according to manufacturer's instructions. Brie y, we extracted cellular and tissue chromatin DNA and digested to approximately 150-700 bp using a micrococcal nuclease (CST). The fragmented DNA was immunoprecipitated with anti-5-mC (Abcam, ab10805) at room temperature for 2 h. After washing and purifying the DNA, we quanti ed the methylation status using qPCR. Primers are list in Additional table S2. m6A-RNA Immunoprecipitation (m6A-RIP) We extracted total RNA from treated A549 cells or tumor tissues, and incubated with DNase according to the TURBO DNA-free TM Kit (ThermoFisher) protocol to avoid DNA contamination. Then, we chemically fragmented 1 µg/µl RNA into ~ 100nt size and incubated with m6A antibody to immunoprecipitate according to the standard protocol for the EpiMark N6-Methyladenosine Enrichment Kit (NEB). Enrichment of m6A containing mRNA was then analyzed using qRT-PCR. Primers targeting m6A-enriched regions of SOX2, SMAD7, and MYC are listed in Additional table S2.

Cell proliferation and migration assay
For cell colony formation assay, A549 or H1792 cells were transfected with siRNA or pcDNA for 24-h and seeded into 6-well plates (500/well). After 1 week, we xed formed colonies and stained with 0.1% crystal violet in 20% methanol, and counted colonies consisting of at least 50 cells. We calculated relative cloning numbers and created plots using GraphPad Prism 7.0 (GraphPad Software, Inc.). For the migration assay, we re-suspended 5 × 10 5 cells in Opti-MEM Reduced Serum Media (Invitrogen) and seeded into the upper chamber of a transwell apparatus (0.8 µm, BD Biosciences), and added complete medium to the bottom chamber to provide chemoattractants for migration. After 24 h, we gently wiped away cells remaining on the upper side of the membrane, and xed cells that had migrated to the lower side of the membranes with methanol and stained with 1% crystal violet. To count migrated cells, we captured images of stained cells in ve random elds by using an optical microscope (Olympus, Japan) and counted samples in triplicate.

Statistics
All experiments were repeated at least three times, unless otherwise stated in the gure legend. We performed statistical analyses using the SPSS v20.0 software (SPSS Inc., Chicago, IL), and used Student's t-test (two-tailed) or one-way ANOVA analysis followed by Tukey's, Sidak's, or Bonferroni test to assess statistical signi cance between or among groups. The relationship between ALKBH5 mRNA expression and DNA methylation, as well as between ALKBH5 and LKB1 mRNA expression, were analyzed by Pearson correlation coe cient. We calculated the survival rate using the log-rank (Mantel-Cox) test. Normality was assumed and variance was compared between or among groups. All numerical data were presented as mean ± standard deviation (SD) and a p-value of < 0.05 was considered signi cant.

Results
Reduced m6A level was associated with aggressive KL lung cancer We rst investigated the association between m6A RNA modi cation and aggressive KL lung cancer. IHC staining showed the reduced m6A level by loss of LKB1 in patients with Kras mutation. And, the lowest of m6A level was found in KL tumor tissues when compared to wild type (WT), L, or K tumor tissues (Fig. 1A, B). The m6A level was inversely correlated with TNM (Tumor, Node and Metastasis) and clinical stage, whereas positively with tumor differentiation in Kras mutant patients, but not in those with Kras wildtype (Fig. 1C). In addition, we found lower m6A level in K specimens that were null for Thyroid transcription factor 1 (TTF1), a positive prognostic feature (Fig. 1D) [30]. Thus, reduced m6A modi cation is associated with aggressive KL lung cancer.
Loss of LKB1 enhanced ALKBH5 responses for m6A reduction in K lung cancer To investigate how m6A is regulated in KL lung cancer, we compared the expression of proteins that act as the m6A writer complex (METTL3, METTL14, and WTAP) and erasers (ALKBH5 and FTO). IHC staining showed that only ALKBH5 expression was higher in KL than that of in K tumor tissues ( Fig. 2A, B). ALKBH5 protein expression was negatively correlated with m6A level, in contrast to positive correlation with the TNM and clinical stage in K lung cancer patients (Fig. 2C). However, LKB1 loss alone was not su cient to change m6A level or ALKBH5 expression or their relationship with aggressive tumor phenotypes (Additional Fig S1 A-D).
To con rm observations of clinical specimens, we screened for the differential expressions of m6A mediators and readers based on database queries. Analysis of TCGA, MTB [8,31], and CCLE databases revealed that ALKBH5 mRNA expression was consistently higher in KL cancer tissues or cells, and negatively correlated with LKB1 expression (Fig. 2D, Additional Fig. S2A-D). Furthermore, elevated ALKBH5 expression correlated with poor prognosis for patients with KRAS mutation (Fig. 2E). Additionally, ALKBH5 had the highest basal mRNA expression in human and mouse lung cancer tissues and cell lines (Additional Fig.S2A-C). Taken together, these ndings indicate that ALKBH5, a major regulator of m6A modi cation, contributes to aggressive phenotypes of KL lung tumors.
To investigate whether LKB1 loss affected ALKBH5 and m6A modi cation, we screened several lung cancer cell lines and categorized them based on KRAS mutation and LKB1 expression status. A549 KL (KRAS mut /LKB1 loss ), H1792 K (KRAS mut /LKB1 high ), H1299 (KRAS wt /LKB1 high ) and H1703 (KRAS wt /LKB1 low ) were selected from each category for further analyses (Additional Fig. S3A). We then generated the loss of LKB1 function by siRNA-LKB1 transfection in H1299 and H1792 cells, while gain-offunction by LKB1 overexpression in H1703 and A549 cells. As expected, LKB1 expression negatively regulated m6A levels in cells with KRAS mutation, but not with KRAS wildtype (Fig. 2F). Consistent with clinical data, western blot and qRT-PCR assays showed that ALKBH5 was also the only modulator negatively regulated by LKB1 in KRAS mut cells, but was unaffected in KRAS wt cells (Fig. 2G, Additional Fig  S3B-E). These observations were supported by immuno uorescence staining that demonstrated negligible ALKBH5 signal in LKB1-overexpression A549 cells (Fig. 2H). Exogenous ALKBH5 expression blocked m6A staining in the presence of LKB1 (Fig. 2I). Furthermore, LKB1 expression negatively correlated with ALKBH5 expression was also found in KRAS-mutated pancreatic and colorectal cancer cell lines (Additional Fig. S4A-D). Therefore, KRAS mutation and LKB1 expression directly affected global m6A levels via ALKBH5.
ALKBH5 upregulation increased cell proliferation and migration in KL lung cancer cells Next, we explored functional relationships between LKB1 and ALKBH5 in KRAS mutant lung cancer cells. First, we established the transient ALKBH5 and/or LKB1 knockdown models in H1792 cells, as well as overexpression in A549 cells (Fig. 3A). As expected, ALKBH5 could fully release LKB1 repressed m6A levels in both cell lines (Fig. 3B, C). Functionally, ALKBH5 knockdown signi cantly inhibited cell proliferation, as shown by decreased colony formation in LKB1-silenced H1792 cells (Fig. 3D). We observed similar for H1792 cell migration in the transwell assay (Fig. 3E). Conversely, ALKBH5 overexpression promoted cell proliferation and migration in LKB1-transfected A549 cells (Fig. 3F, G). Thus, LKB1 loss correlated with upregulation of ALKBH5, and increased cell proliferation and migration in KRAS mutant cells.
Loss of LKB1 upregulated ALKBH5 via DNA hypermethylation in KRAS mutant cancer cells.
We then explored how LKB1 regulated ALKBH5 through DNA methylation. Consistent with previous studies in pancreatic ductal epithelial cells [11], LKB1 alterations also negatively regulated the global 5mC DNA methylation in KRAS mutant lung cancer cells (Fig. 4A) but had no effect in KRAS wildtype cells. Speci cally, ALKBH5 gene DNA methylation positively correlated with ALKBH5 mRNA expression in KRAS mutant lung, and colorectal cancer cell lines, based on the CLLE database (Additional Fig. S5A-D).
Subsequent treatment with 5-azacytidine (5-aza), an inhibitor of DNA methylation, reduced ALKBH5 mRNA and protein expression in a dose-dependent manner in H1792 and A549 cells, independent of LKB1 status (Fig. 4B-D). Based on ChIP-sequence analysis from the ENCODE database, we identi ed a single putative transcriptional repressor, CTCF (CCCTC-binding factor) and several activators, including H3K4me1, H3K4me2, H3K4me3, H3K9ac, and H3K27ac, on the ALKBH5 gene core promoter in A549 cells (Additional Fig. S6). Interestingly, the CTCF peak region was co-located in the CpG island of ALKBH5 promoter (Fig. 4E). Further analysis by MeDIP assay indicated 5mC-methylation on the CTCF peak region was decreased with 5-aza treatment or exogenous LKB1-transfection in A549 cells (Fig. 4F). Lastly, bisul te sequencing con rmed decreased DNA methylation on the CTCF peak region in 5-aza-treated or LKB1 over-expressed cells (Fig. 4G, H). Thus, the data indicates that LKB1 loss induced DNA hypermethylation, thereby controlling ALKBH5 expression in KRAS mutant cancer cells.
DNA hypermethylation of the CTCF motif is critical for ALKBH5 upregulation.
To determine whether CTCF directly represses ALKBH5, we used western blot and RT-qPCR. We found that silencing CTCF increased ALKBH5 protein and mRNA expression, and also rescued ALKBH5 repression by LKB1 overexpression (Fig. 5A-C). We next generated serial deletions, including CTCF motif deletion constructs based on the human ALKBH5 promoter, for the luciferase reporter assay. The CTCF-containing construct had lower ALKBH5 promoter activity than constructs that lacked the motif (Fig. 5D). Notably, CTCF motif deletion increased ALKBH5 promoter activity to levels similar to those in constructs without the CTCF motif. This evidence suggests that CTCF directly represses ALKBH5 transcriptional activity.
Next, we found that DNA demethylation by 5-aza treatment or by LKB1 overexpression reduced ALKBH5 promoter activity on the CTCF-containing construct, but had no effect on CTCF-de cient construct (Fig. 5E). Further ChIP-qPCR analysis indicated that histone activators, such as H3K4me1, H3K4me2, H3K4me3, H3K9ac, and H3K27ac, occupy the ALKBH5 gene promoter (Fig. 5F), consistent with ChIP-seq results (Additional Fig. S6). Interestingly, 5-aza treatment or LKB1 overexpression promoted CTCF enrichment on ALKBH5, and also partially prevented enrichment of H3K4me3, H3K9ac, and H3K27ac ( Fig. 5H-J). Therefore, loss of LKB1 promoted DNA hyper-methylation in the CTCF peak region, thus preventing CTCF binding and releasing repression of ALKBH5 in KRAS mutant cells.
ALKBH5 demethylation of m6A increased expression and stability of SOX2, SMAD7, and MYC in a YTHDF2-dependent pathway To identify downstream targets of ALKBH5 mediated m6A modi cation in lung cancer, we overlapped 2,605 genes using the canonical m6A motif-enriched gene stop codon region based on m6A-sequence in A549 cells (20) (Additional Fig. S7A). Gene ontology analysis revealed that the 2,605 genes were signi cantly enriched in gene transcription regulation, mRNA splicing, cell cycle, and mRNA stability (Additional Fig. S7B). KEGG pathway analysis revealed that the overlapping genes were closely associated with Hippo and TGF-β pathways (Additional Fig. S7C). Using qRT-PCR, we con rmed that 45.2% (14/31) of Hippo-Yap pathway genes were directly regulated by ALKBH5 (Additional Fig. S7D, E). Western blot assay further con rmed that ALKBH5 regulated SOX2, SMAD7, and MYC proteins in A549 and H1792 cells (Fig. 6A, B).
To investigate m6A modi cations on SOX2, SMAD7, and c-MYC mRNA, we used re y luciferase assay (Fig. 6C). We found their activities were signi cantly less than those of their mutant reporters, indicating that m6A repressed gene activity. Moreover, overexpression of ALKBH5 increased luciferase activities of three m6A WT gene reporters, and also rescued the repressive activity mediated by LKB1-transfection.
However, we saw no such effects for m6A mutant gene-fused reporters. Our m6A-RIP-qPCR assay showed signi cantly higher accumulation of m6A at mRNA fragments of all three genes than for the IgG control. However, this m6A enrichment was reduced by ALKBH5 overexpression. Also, ALKBH5 upregulation inhibited the increased m6A occupancy caused by LKB1 overexpression (Fig. 6D). Given that ALKBH5 overexpression or knockdown did not change the pre-mRNAs of SMAD7 and MYC (Additional Fig S8A-D), we predicted m6A reader protein YTHDF2 regulated their mRNA stability. Interestingly, actinomycin-D induced degradation of SMAD7, SOX2, and MYC mRNAs was partially prevented by silencing YTHDF2 in both A549 and H1792 cells. (Additional Fig. S8E-J). RIP-qPCR assay showed that YTHDF2 also occupied SMAD7, SOX2, and MYC m6A regions, and this enrichment increased in the absence of ALKBH5 (Fig. 6E). Furthermore, YTHDF2 silencing rescued their gene expression upon LKB1 overexpression or ALKBH5 knockdown (Fig. 6F). Thus, ALKBH5-m6A-YTHDF2 signaling prevented SOX2, SMAD7, and MYC mRNA decay.
Next, we validated the underlying regulation of m6A by LKB1 using a panel of clinically relevant lung adenocarcinoma specimens. LKB1 protein expression negatively correlated with global 5-mC DNA methylation in lung cancer tissues with KRAS mutations, but not in tissues with WT KRAS (Additional Fig.  S9A, B). Notably, ALKBH5 DNA methylation positively correlated with ALKBH5 mRNA expression and both expression signi cantly increased with LKB1 de ciency in patients with KRAS mutation (Additional Fig.  S9C-E). ALKBH5 protein expression negatively correlated with global m6A level, as well as the m6A levels for SOX2, SMAD7, and MYC genes in KRAS mutant tissues (Additional Fig. S9F-H). Furthermore, m6A levels of these three genes negatively correlated with their mRNA expression (Additional Fig. S9I-K).

Discussion
Here, we report that reduced m6A RNA modi cation increased stability and expression of critical oncogenes and contributed to aggressive cancer phenotypes. Loss of LKB1 speci cally enhanced ALKBH5 expression and reduced m6A levels in KRAS mutated cells. LKB1 inactivation could increase the 5-mC DNA methylation of the ALKBH5 promoter, which prevents CTCF binding and releases ALKBH5 suppression. Subsequently, reduced "m6A-YTHDF2" signaling promoted the expression and stability of critical tumor oncogenes, such as SOX2, SMAD7, and MYC. Our ndings describe a mechanism of crosstalk between 5mC-DNA and m6A RNA modi cation in KL tumors that supports tumorigenic growth and progression (Fig. 7). Our study indicates that LKB1 loss reduced m6A modi cation by upregulating ALKBH5, which contributes to aggressive tumor progression and poorer outcomes for KL lung cancer.
Aberrant global m6A abundance is increasingly reported in human cancers, and may be associated with cancer progression and clinical outcome [32]. Interestingly, m6A hypomethylation was reported in glioblastoma, bladder cancer, endometrial cancer [33], melanoma, or breast carcinoma, whereas, m6A hypermethylation was found in gastric cancer and hepatocellular carcinoma [34]. Writing and erasing proteins regulate m6A levels, which enable the binding of m6A reader proteins and initiate a series of biological functions. For example, ALKBH5 demethylates m6A mRNAs, and modulates mRNA splicing, export and stability. Alkbh5 de ciency leads to aberrant spermatogenesis and apoptosis in mouse testes, likely through regulating genes associated with the p53 network [22]. ALKBH5 often exerts an oncogenic role in GBM, pancreas, cervical, and breast cancer, but acts a tumor-suppressor in leukemia [32]. This twosided role of ALKBH5, might relate with the complex and diverse function of m6A modi cation, which not only promotes the translation of related mRNAs but also reduces the mRNAs stability by binding with different reader proteins [13][14][15][16]. Thus, It would be important to determine how critical role of m6A and its regulator in each type of cancer.
Limited literature is available to explain how m6A regulators modulate cancers, although many studies have describes roles for m6A regulators in cell fate and carcinogenesis [20,35,36]. This study identi es ALKBH5 as a functional target gene of 5-mC DNA, which controls m6A RNA. Notably, ALKBH5 is one of the top ve 5-mC DNA biomarkers that helped distinguish patients with metastatic-lethal prostate cancer [37], suggesting that DNA methylation of ALKBH5 may be a prognostic indicator.
We also identi ed CTCF as an effective transcriptional suppressor that was highly sensitive to global 5-mC DNA methylation and required for ALKBH5 repression. Similarly, we previously showed that DNA hypomethylation facilitated CTCF binding to and suppression of hTERT expression in human endothelium [26]. Recent studies show 5-mC DNA hypermethylation facilitated tumorigenesis in KRAS/LKB1 co-mutated cancer [11]. LKB1 inactivation causes DNA hypermethylation and histone methylation, which facilitates immune escape in KL-mutated lung cancer and represses anti-oncogenic STING [38]. Consistent with those previous ndings, our work indicates that oncogenic ALKBH5 is another target of LKB1 by a similar mechanism. Thus, loss of LKB1 reduces m6A modi cation also might via the linking of 5mC-DNA and histone modi cation in KL cancer.
Consistent with previous studies, our observations indicate that ALKBH5 is a lung cancer oncogene because it promoted cell proliferation and migration [18,21]. Increased ALKBH5 expression predicted poor survival in our analysis. Moreover, we identi ed three critical oncogenes, SOX2, SMAD7, and MYC, as targets of ALKBH5-mediated m6A modi cation. Mechanistically, LKB1 loss or ALKBH5 overexpression increased SAMD7, SOX2, and MYC stability via a YTHDF2-dependent mechanism. This result is supported by previously reported PAR-CLIP-Seq data in a Hela cell line [13]. YTHDF2-induced decay of m6A modi ed genes may be a common pathway across cell types. We also found that YTHDF1 occupied and promoted SAMD7 or MYC mRNA translation in an m6A-dependent manner [39]. Thus, it would be interesting to compare proteomic changes between tumor and non-tumor samples upon METTL3 manipulation.
Notably, LKB1 regulation of ALKBH5 via 5mC-DNA was not limited to KRAS mutant lung cancer, but extended to pancreatic and colorectal cancer, which are the top three causes of cancer death in the United States [40]. Aggressive lung tumorigenesis, tumor progression, and poor prognosis were observed in mice with Kras mutation combined with Lkb1 inactivation [8]. This tumor type is largely resistant to both standard-of-care treatments like docetaxel and combination treatment with a MEK inhibitor [5,41]. Comutations were also associated with an inert tumor immune microenvironment and poor clinical response to immune checkpoint blockade [42][43][44]. Thus, understanding molecular mechanisms may improve therapeutic strategies for cancer with KRAS and LKB1 co-mutations. Screening chemicals that may regulate m6A formation or removal is an effective approach for developing tumor therapeutics. For example, R-2HG, an ALKBH5 and FOT inhibitor, inhibits leukemia cell growth and induces apoptosis in mice [45]. m6A-YTHDF2 inactivity contributes to melanoma progression by enhancing the expression and stability of key immune checkpoint factors, including PD-1, CXCR4, and SOX10 [46]. This implies that YTHDF2 modulation could be combined with an anti-PD-1/PD-L1 blockade to improve anticancer immunotherapy. In support of this idea, we identi ed the ALKBH5-m6A-YHTDF2 axis as a targetable molecular pathway to treat this aggressive cancer.

Conclusions
In conclusion, our results suggest that LKB1 loss caused epigenetic reprogramming via 5mC-DNA methylation. We also found that histone modi cation was necessary for ALKBH5 upregulation, which in turn controlled m6A RNA modi cation for KRAS co-mutated cancers. Our ndings and other recent reports [10,14,15,17] highlight the importance of the global m6A modi cation in cancer. Global m6A modi cation may selectively activate oncogenic drivers, such as SOX2, SMAD7, and MYC, to promote aggressive tumor phenotypes. Our ndings on epigenetic reprogramming indicate new therapeutic approaches to tumors with dual LKB1 and KRAS mutation.          reporter assay of A549 cells transfected with pGL3-basic constructs containing serial LKBH5 promoters or deletion of CTCF peak fragment. n = 8/group, mean ± SD. * P < 0.05 by one-way ANOVA followed by Tukey's test. (E) Reduction of ALKBH5 Luc:-1168bp wildtype (WT) construct activities by treatment of 5aza or LKB1 over-expression in A549 cells, but not for ALKBH5 Luc:-1168bp deletion (Del). Data as mean±SD (n=5), *P < 0.05 (Student's t test). (F) ChIP-qPCR showing that the ALKBH5-CTCF peak region was occupied by the suppressor of CTCF and activators of histone modulators in A549 cells. (G-J) ChIP-qPCR analyses of 5aza treated or LKB1 over-expressed A549 cells. n = 3/group, mean ± SD. *P< 0.05 vs.
IgG in F, vs. A549-DMSO in G-J by one-way ANOVA followed by Tukey's test.

Figure 5
Suppressor-CTCF is required for ALKBH5 down-regulation by LKB1. (A-C) Representative images and quanti cation of CTCF and ALKBH5 protein expression by WB and qRT-PCR in A549 cells with LKB1 overexpression and/or CTCF knockdown for 48hrs. Error bars, SD (n = 4 for WB; n=5 for qRT-PCR), *P < 0.05 vs. SI-CN or OE-CN (2-way ANOVA with Bonferroni multiple comparison post hoc test). (D) Luciferase reporter assay of A549 cells transfected with pGL3-basic constructs containing serial LKBH5 promoters or deletion of CTCF peak fragment. n = 8/group, mean ± SD. * P < 0.05 by one-way ANOVA followed by Tukey's test. (E) Reduction of ALKBH5 Luc:-1168bp wildtype (WT) construct activities by treatment of 5aza or LKB1 over-expression in A549 cells, but not for ALKBH5 Luc:-1168bp deletion (Del). Data as mean±SD (n=5), *P < 0.05 (Student's t test). (F) ChIP-qPCR showing that the ALKBH5-CTCF peak region was occupied by the suppressor of CTCF and activators of histone modulators in A549 cells. (G-J) ChIP-qPCR analyses of 5aza treated or LKB1 over-expressed A549 cells. n = 3/group, mean ± SD. *P< 0.05 vs.
IgG in F, vs. A549-DMSO in G-J by one-way ANOVA followed by Tukey's test.  Proposed model for LKB1-mediated m6A modi cation in KRAS mutated lung cancer progression. Loss of LKB1-induced DNA hypermethylation, which prevents CTCF binding on the ALKBH5 gene promoter, maintains ALKBH5 expression, and further represses global RNA methylation. Oncogenic SMAD7, SOX2, and MYC are crucial targets of m6A meditated by LKB1 de ciency, and are involved in KRAS mutation lung cancer progression.

Figure 7
Proposed model for LKB1-mediated m6A modi cation in KRAS mutated lung cancer progression. Loss of LKB1-induced DNA hypermethylation, which prevents CTCF binding on the ALKBH5 gene promoter, maintains ALKBH5 expression, and further represses global RNA methylation. Oncogenic SMAD7, SOX2, and MYC are crucial targets of m6A meditated by LKB1 de ciency, and are involved in KRAS mutation lung cancer progression.

Supplementary Files
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