Menin is necessary for the formation of the menin/mixed lineage leukemia (MLL) complex and is recruited directly to chromatin. Menin is an important tumor suppressor in several cancer types, including lung cancer. Here, we investigated the role of MLL in menin-regulated lung tumorigenesis. Ablation of MLL suppressed KrasG12D-induced lung tumorigenesis in a genetically engineered mouse model. MLL deficiency decreased histone H3 lysine 4 trimethylation (H3K4me3) and subsequently suppressed expression of the Ras protein-specific guanine nucleotide-releasing factor 1 (Rasgrf1) gene. Rasgrf1 was essential for the GTP-bound active state of Kras and the activation of Kras downstream pathways as well as their cancer-promoting activities. MI-3, a small-molecule inhibitor targeting MLL, specifically inhibited the growth of Kras-mutated lung cancer cells in vitro and in vivo with minimal effect on wild-type Kras lung cancer growth. Together, these results demonstrate a novel tumor promoter function of MLL in mutant Kras-induced lung tumorigenesis and further indicate that specific blockade of the MLL-Rasgrf1 pathway may be a potential therapeutic strategy for the treatment of tumors containing Kras mutations.

Significance:

Activation of mutant Kras is dependent on MLL-mediated epigenetic regulation of Rasgrf1, conferring sensitivity to small-molecule inhibition of MLL in Kras-driven lung cancer.

Tumorigenesis is currently recognized as the result of the synergistic effect of genetics and epigenetics. The Kras gene is one of the most frequently mutated oncogenes in various cancers (1). Ras proteins are membrane-bound small GTPases that have GTP-bound active states and GDP-bound inactive states that are regulated by Ras guanine nucleotide exchange factors (Ras-GEF) and Ras GTPase-activating proteins (Ras-GAP), respectively (2). Most Kras mutations are single-base missense mutations, often occurring at codons 12, 13, or 61, which inhibit the hydrolysis of GTP that is normally enhanced by Ras-GAPs and accelerate the activation of downstream signaling pathways such as the MAPK and PI3K pathways (3). In lung adenocarcinoma (LUAD), pancreatic ductal adenocarcinomas, and colorectal carcinomas, in which Kras mutations predominate, over 75% of amino acid substitutions occur at the G12 codon position (3). The expression of mutant KrasG12D in alveolar type 2 cells (ATII) and Clara cells in mouse lungs can give rise to LUAD (4). Recently, mounting evidence has shown an intimate relationship between epigenetic alterations and the pathogenesis of lung cancer. It has been demonstrated that nuclear receptor-binding SET domain-containing 2 (NSD2) cooperates with oncogenic Kras signaling via histone H3 lysine 36 dimethylation (H3K36me2) to drive LUAD tumorigenesis (5). YEATS2 is a histone 3 lysine 27 acetylation reader that regulates a transcriptional program for non–small cell lung cancer (NSCLC) tumorigenesis (6). Mixed lineage leukemia 2 (MLL2) deficiency promotes lung tumorigenesis by impairing epigenomic signals for enhancers, including superenhancers for the tumor suppressor Per2, which regulates multiple glycolytic genes (7).

The MLL1 (MLL) gene encodes a methyltransferase that is involved in the trimethylation of histone 3 lysine 4 (H3K4me3) and positively regulates the expression of multiple genes (8). As an epigenetic regulator, MLL plays a vital role in many tumors, including leukemia, cervical tumors, and pancreatic cancer (9). Menin is encoded by the multiple endocrine neoplasia (MEN1) gene; menin is necessary for the formation of the menin/MLL complex and is recruited directly to the chromosome (10). Menin/MLL regulates the expression of P18 and P27 through H3K4me3, and menin inactivation leads to the reduced expression of P18 and P27, which are related to islet tumorigenesis (11, 12). Our research has demonstrated that menin is an important tumor suppressor in lung cancer (13–16). Importantly, loss of MEN1 results in spontaneous mixed-type lung cancer in mice, dramatically accelerates KrasG12D-induced lung tumors and leads to neuroendocrine (NE) differentiation of lung cancer (16).

Here, we hypothesized that MLL participates in menin-regulated lung tumorigenesis and NE differentiation. Surprisingly, using genetically engineered mouse models (GEMM), we found that MLL ablation suppresses KrasG12D-induced lung tumorigenesis, and the mechanism was related to the MLL-induced epigenetic regulation of the Rasgrf1. A small-molecule inhibitor targeting MLL specifically suppressed lung cancer cells harboring Kras mutants in vitro or in vivo. However, ablation of MLL promoted NE differentiation of LUAD cells. Collectively, our results demonstrate a novel tumor-promoting function of MLL in mutant Kras-induced LUAD tumorigenesis that is independent of menin.

Ethics and mice

We conducted all animal experiments in strict accordance with animal welfare and other related ethical regulations, and the procedures were approved by the Institutional Animal Care Committee of the Medical College at Xiamen University (Xiamen, Fujian, P.R. China). All mice were housed in a light- and temperature-controlled sterile room where humidity was carefully monitored and controlled. The protocols for LSL-KrasG12D/+ mice, MLLf/f mice, Sftpc-Cre mice, and UBC-cre mice have been described previously (16), and Scgb1a1-Cre mice were obtained from Jackson Laboratory. All animals were maintained on a C57BL/6 background, and equal proportions of males and females were used in all experiments (genotyping primers listed in Supplementary Table S2). At 6–8 weeks of age, mice received intraperitoneal injections of 100 mg/kg tamoxifen (TAM) as described previously (16). At 3 or 5 months following injection or at the ethical endpoint, lungs were harvested for further analysis. For survival studies, as reported previously (16), the endpoints included but were not limited to labored and fast breathing, reduced eating or movement, energy loss, or weight loss > 20% of the initial body weight.

Cell culture

A549 cells were maintained in Ham's F-12K (BasalMedia), NCI-H1299 cells were maintained in DMEM (BasalMedia), NCI-H1944, NCI-H157, and NCI-H1975 cells were maintained in RPMI1640 medium (BasalMedia), additional material such as Hepes (Hyclone), NEAA (Hyclone), sodium pyruvate (Hyclone), and l-glutamate (Hyclone) are added as needed. The F12K, DMEM, and RPMI1640 contained 10% FBS (Cegrogen), and 1% pen/strep (Invitrogen). The cell lines were cultured at 37°C in an atmosphere of 5% CO2. A549, NCI-H1944, NCI-H157, and NCI-H1975 were obtained from National Collection of Authenticated Cell Cultures, and NCI-H1299 was obtained from ATCC, all cells were used within 6 months. Mycoplasma infection was routinely checked by a Myco-Lumi Luminescent Mycoplasma Detection Kit (Beyotime).

Isolation of primary MEF cells

LSL-KrasG12D/+ mice crossed with UBC-cre mice to generate LSL-KrasG12D/+; UBC-cre mice, LSL-KrasG12D/+; MLLf/f mice crossed with MLLf/f; UBC-cre mice to generate LSL-KrasG12D/+; MLLf/f; UBC-cre mice. The wild-type (WT) cells (LSL-KrasG12D/+ cells, MLLf/f cels and LSL-KrasG12D/+; MLLf/f cells) were controls. And the following steps were performed as described previously (17). The cells were used in 3–4 passages, and were tested negative for Mycoplasma.

Isolation of ATII cells

Isolation of ATII cells was performed according to an article (18) with some modifications. Treated mice were sacrificed by CO2 asphyxiation. Spray the mouse with 75% ethanol and then the thoracic cavity was opened. Exsanguinate the mice by cutting the left and right jugular vein as well as the renal artery. Exposure of the trachea and take care not to injure the lung. To use a 10 mL syringe filled with cold PBS, puncture the right ventricle of the heart and perfuse the lung with PBS until free of blood. A 22G indwelling needle is inserted into the trachea and connected to the lung. A total of 1.5 mL of dispase II was instilled into the lungs and followed by 0.2 mL of 1% low melting point agarose. The lungs were immediately covered with ice and incubated for 2 minutes. The lungs were isolated and incubated in 2 mL of dispase II for 45 minutes at room temperature. Then, the lungs were minced and a transfer pipette in DMEM containing antibiotics. After mincing, 100 μL of 1 mg/mL DNase I was added to the sample and incubated for 4 minutes at room temperature. The cells were sequentially filtered through 100, 70, and 30 μm filters. Next, the cell pellet was suspended with 1 mL of erythrocyte lysis buffer and incubated for 1 minute at room temperature, followed by washing with cold PBS. Incubate the cells with antibodies for 10–30 minutes in the dark at 4°C. Resuspend the cells in DMEM at dilutions suitable for flow cytometry.

Tumorigenesis assay in vivo

A total of 3 × 106 A549 or 1 × 107 H1299 (containing vector or KrasG12V plasmid) human LUAD cells were injected subcutaneously into the flanks of 6–8 weeks male nude mice as described previously (16). Mice were examined every 2–3 days for signs of tumor growth and behavior. Once the tumor size reached 100–200 mm3 (about 3–4 weeks after injection cells), 15 mg/kg MI-3 (in ddH2O containing 10% DMSO, 30% PEG300, and 10% Tween 80) was intraperitoneally injected every 2–4 days for 2 weeks, control mice were injected with equal volume of solvent. The mice were euthanized after MI-3 treated for 2 weeks, and the xenograft tumors were dissected for analysis.

Ras activity assay

After treatment, cells were grown to 70% confluence in a 60-mm dish, starved overnight in serum-free medium, and allowed to recover in 10% FBS medium for 20 minutes. The activity of Ras was detected according to the manufacturer's instructions.

Chromatin immunoprecipitation assays

Chromatin immunoprecipitation (ChIP) assays were performed as described previously (16), and the antibodies and primer pair (PP) sequences for the ChIP assays are shown in Supplementary Table S2.

siRNAs targeting MLL and Rasgrf1

A total of 50 nmol/L siRNAs were introduced into cells according to the transfection protocol. The sequences of siRNAs are listed in Supplementary Table S2.

Establishment of KrasG12V stably overexpressing cell line

Lenti-mCherry-G12V-Kras plasmid was purchased from Addgene and used to generate stable cell lines. To produce KrasG12V-overexpressing cell lines, H1299 or H157 cells were infected with lentiviral particles and the cells with mCherry were sorted by flow cytometry. Cells infected with lentiviral expressing an empty vector plasmid were used as controls.

Histology and IHC

Lungs were isolated and fixed in 4% paraformaldehyde for 48 hours at 4°C and embedded in paraffin. Five-μm–thick sections were stained with hematoxylin and eosin (H&E) and immunostaining, then the sections were photographed and quantitative analysis as described previously (16). Primary antibodies were listed in Supplementary Table S1.

Study of human primary lung cancer specimens

These studies were approved by the Medical Ethics Committee of Xiamen University (Xiamen, Fujian, P.R. China) and Jilin University (Changchun, P.R. China). The demographic data and clinicopathological features of the patients with NSCLC are listed in a previous report (16).

RNA sequencing and bioinformatics analysis

The MLLf/f and MLLf/f; UBC-cre mice (n = 3 for per group) received intraperitoneal injections of TAM, and the genotype was verified by PCR 1 week later. After 4 months, the lung tissues were removed and sent to the manufacturer (Novogene) for RNA sequencing (RNA-seq). Quality of RNA samples was determined by agarose gel electrophoresis, NanoPhotometer spectrophotometer, Qubit 2.0 Fluorometer, and Agilent 2100 bioanalyzer, and the RNA-seq libraries were sequenced using Illumina HiSeq Xten. The raw data were available in NCBI (PRJNA830337). Differential gene expression analysis was performed with the DESeq2 package, genes with Padj < 0.05 as the difference significance criterion. Gene set enrichment analysis (GSEA) was performed at AMOGENE (Xia Men). Genes upregulated by KrasG12V expressed data were obtain from Gene Expression Omnibus series GSE65258. MLL, Rasgrf1, and Chk1 mRNA data were obtained from human LUAD The Cancer Genome Atlas (TCGA) database. The MLL mRNA expression data of LUAD, liver hepatocellular carcinoma (LIHC), colon adenocarcinoma (COAD), pancreatic adenocarcinoma (PAAD), and prostate adenocarcinoma (PARD) were obtained from TCGA database.

Data availability

The data generated in this study are available upon request from the corresponding author.

Quantification and statistical analysis

Statistical analysis was performed using Prism software (GraphPad Prism 8). Survival analyses were performed with the log-rank (Mantel–Cox) test. Statistical analyses of qPCR, colony formation, and IHC data were performed with two-tailed unpaired t tests. Spearman correlation coefficients were calculated using Spearman test.

Ablation of MLL attenuates KrasG12D-driven LUAD

MEN1 deficiency dramatically accelerates KrasG12D-induced lung tumorigenesis and promotes NE differentiation (16). Given that the menin/MLL complex controls both gene transcriptional regulation and disease pathogenesis (19), we hypothesized that MLL participates in menin-regulated lung tumorigenesis. Two GEMMs with ATII (Sftpc-Cre) and Clara (Scgb1a1-Cre) cell–specific KrasG12D activation concomitant with MLL ablation were developed (Supplementary Fig. S1A). The successful generation of mice with the KrasG12D/+ allele and deletion of the MLL allele was detected by PCR using DNA isolated from lung tissues of LSL-KrasG12D/+;Scgb1a1-Cre (KSc) and LSL-KrasG12D/+;MLLf/f;Scgb1a1-Cre (KMSc) mice (Supplementary Fig. S1B). The KSc mice began to die 20 weeks after intraperitoneal injection of TAM, while the KMSc mice started to die beyond 30 weeks, mice were killed when they became moribund. MLL deletion moderately prolonged survival in mice with Scgb1a1-cell–specific KrasG12D mutation (Fig. 1A). The lung tissues of mice were examined morphologically and pathologically. Consistent with a previous report (4), the majority of nodules and tumors arose in the alveolar compartment at the lung periphery in ATII cell-specific KrasG12D-mutant mice, while they were observed in close proximity to the bronchioalveolar duct junction in Clara cell–specific KrasG12D-mutant mice (Fig. 1B). Both KSc and LSL-KrasG12D/+;Sftpc-Cre (KSf) mice developed macroscopic tumors, while the tumors in both KMSc and LSL-KrasG12D/+;MLLf/f;Sftpc-Cre (KMSf) mice were significantly reduced (Fig. 1B), the lung tissue of MLL knockout (KO) mice (MSf, MLLf/f;Sftpc-Cre and MSc, MLLf/f;Scgb1a1-Cre) were normal. The lung coefficient (lung/body weight) was significantly reduced in the KMSc mice compared with the KSc mice (Fig. 1C). H&E staining and tumor burden analysis also indicated that MLL ablation inhibited KrasG12D-driven lung tumorigenesis in the two GEMMs (Fig. 1B and D). All groups showed TTF1 immunostaining, but there was no difference among them, indicating that the tumors were primary lung cancers (Fig. 1E). The tumors in mice lacking MLL showed lower proliferation, as determined by Ki67 immunostaining (Fig. 1E and F). However, MLL deletion resulted in upregulation of neural cell adhesion protein 1 (NCAM1), an NE marker, confirming that MLL is also involved in regulating NE differentiation (Fig. 1E and G). In contrast to the finding that MEN1 deficiency recovered Kras-induced epithelial–mesenchymal transition (EMT) in lung tumors (16), MLL ablation did not affect EMT, which was determined by E-cadherin and vimentin immunostaining (Fig. 1E and H). Together, these results indicate that MLL is an essential promoter of Kras-driven lung tumorigenesis, serving an opposite role to menin, which functions as a tumor suppressor in lung cancer pathogenesis.

Figure 1.

MLL ablation suppresses Kras-driven LUAD. A, Shown are Kaplan–Meier survival curves of KSc (n = 12, median survival = 35 weeks) and KMSc (n = 7, median survival = 41 weeks) mice. The P values were determined by log-rank (Mantel–Cox) test. B, Representative macroscopic lung images with H&E of lung tissue sections from KSf and KMSf mice at 12 weeks; KSc and KMSc mice at 20 weeks after intraperitoneal injection of 100 mg/kg TAM. Scale bars, 3 mm. C, Scatter plot of lung weight/body weight of the indicated mice (WT, n = 8; MSc, n = 17; KSc, n = 17; KMSc, n = 26), MLLf/f;Scgb1a1-Cre (MSc); WT mice as controls. D, Quantification of tumor burden (tumor area per lung) in the indicated mice (KSf, n = 19; KMSf, n = 25; KSc, n = 12; KMSc, n = 26). E, Representative images of lung tissue sections stained with H&E or for Ki67, TTF1, NCAM1, E-cadherin, and vimentin by IHC staining in the KSf (n = 13), KMSf (n = 8), KSc (n = 7), and KMSc (n = 8) mice. Scale bars, 60 μm. FH, Quantification of Ki67, NCAM1, E-cadherin, and vimentin IHC staining in the indicated mice in E. I, Quantification of F4/80 IHC staining shown in Supplementary Fig. S2E (WT, n = 5; MSf, n = 8; KSf, n = 9; KMSf, n = 8). J, qRT-PCR was used to detect the mRNA expression of iNos (M1 type macrophage maker) and Arg (M2-type macrophage maker) in WT (n = 3), KSf (n = 4), and KMSf (n = 5) lung tissues. Data are represented as mean ± SEM in C, D, F, G, H, I, and J, and the P values were determined by two-tailed unpaired t tests.

Figure 1.

MLL ablation suppresses Kras-driven LUAD. A, Shown are Kaplan–Meier survival curves of KSc (n = 12, median survival = 35 weeks) and KMSc (n = 7, median survival = 41 weeks) mice. The P values were determined by log-rank (Mantel–Cox) test. B, Representative macroscopic lung images with H&E of lung tissue sections from KSf and KMSf mice at 12 weeks; KSc and KMSc mice at 20 weeks after intraperitoneal injection of 100 mg/kg TAM. Scale bars, 3 mm. C, Scatter plot of lung weight/body weight of the indicated mice (WT, n = 8; MSc, n = 17; KSc, n = 17; KMSc, n = 26), MLLf/f;Scgb1a1-Cre (MSc); WT mice as controls. D, Quantification of tumor burden (tumor area per lung) in the indicated mice (KSf, n = 19; KMSf, n = 25; KSc, n = 12; KMSc, n = 26). E, Representative images of lung tissue sections stained with H&E or for Ki67, TTF1, NCAM1, E-cadherin, and vimentin by IHC staining in the KSf (n = 13), KMSf (n = 8), KSc (n = 7), and KMSc (n = 8) mice. Scale bars, 60 μm. FH, Quantification of Ki67, NCAM1, E-cadherin, and vimentin IHC staining in the indicated mice in E. I, Quantification of F4/80 IHC staining shown in Supplementary Fig. S2E (WT, n = 5; MSf, n = 8; KSf, n = 9; KMSf, n = 8). J, qRT-PCR was used to detect the mRNA expression of iNos (M1 type macrophage maker) and Arg (M2-type macrophage maker) in WT (n = 3), KSf (n = 4), and KMSf (n = 5) lung tissues. Data are represented as mean ± SEM in C, D, F, G, H, I, and J, and the P values were determined by two-tailed unpaired t tests.

Close modal

Nevertheless, the survival curves and lung coefficients did not differ between KMSf and KSf mice (Supplementary Fig. S1C and S1D). On the basis of the morphologic observations and H&E staining, there were two phenotypes in KMSf mice: 41% of mice showed changes consistent with KMSc mice, with fewer nodules and smaller tumors; 59% of mice showed increased lung volume, ATII cell damage, disorganized lung tissue, collapsed alveoli, many inflammatory cells and plasma cell infiltration and very few nodules (Fig. 1B; Supplementary Fig. S1E). Immunostaining for cleaved caspase-3 indicated that MLL KO increased the number of apoptotic cells compared with that in the control groups (Supplementary Fig. S1E and S1F). Abundant macrophages existed in the lungs of KMSf mice, as indicated by increased F4/80 immunostaining and iNos and Arg mRNA levels (Fig. 1I and J; Supplementary Fig. S1E). Furthermore, CD8+ T-cell infiltration in the lungs of KMSf mice was also increased (Supplementary Fig. S1E and S1G). Morphologic observations revealed that the lung tissue of KMSf mice showed features that were similar to those found in pulmonary alveolar proteinosis (PAP), which is characterized by abnormal air exchange, alveolar surfactant accumulation, and respiratory distress (20). We performed Periodic acid-Schiff (PAS) and Alcian blue staining and found that 59% of KMSf mice without tumors indeed had PAP; however, the KMSc mice did not have a similar phenotype (Supplementary Fig. S1H and S1I). We suspect that MLL is essential for the survival of ATII cells and the maintenance of alveolar structures and that the lack of these factors, and not the tumors, was the primary pathologic cause of death in KMSf mice.

MLL ablation antagonizes Kras pathway activation

To investigate how MLL participates in Kras-induced lung tumorigenesis, we isolated lung tissues from MLLf/f and MLLΔ/Δ mice and compared their gene expression profiles using RNA-seq (PRJNA830337). Bioinformatics analyses of RNA-seq data using GSEA showed enrichment of genes in the Kras, AKT, mTOR, and MAPK pathways (Fig. 2A; Supplementary Fig. S2A). Next, we compared genes downregulated by MLL KO with those upregulated by Kras activation (RNA-seq data, GSE65258) and found 721 common genes (Supplementary Fig. S2B). These overlapping genes were analyzed by Kyoto Encyclopedia of Genes and Genomes pathway analysis, which showed enrichment of the Ras, MAPK, and PI3K-AKT pathways (Supplementary Fig. S2C). To verify the regulation of the Kras pathway by MLL, primary WT, LSL-KrasG12D/+ and LSL-KrasG12D/+;MLLf/f mouse embryonic fibroblasts (MEF) were isolated, and the MEFs were treated with 4-hydroxytamoxifen (4-OH-TAM) to generate WT, LOX-KrasG12D/+ (LK), and LOX-KrasG12D/+;MLLΔ/Δ (LKM) cells, the effects of genetic recombination were showed in Supplementary Fig. S2D. The expression of MLL mRNA in WT, LK, and LKM MEFs was obtained by qRT-PCR (Supplementary Fig. S2E). Interestingly, Kras activation moderately upregulated MLL mRNA expression in MEFs. Ras-GTP activity assays indicated that ablation of MLL inhibited the Ras-GTP activity induced by Kras mutation in primary MEFs (Fig. 2B). Two distinct MLL siRNAs were used to treat A549 (KrasG12S) cells, and the reduced MLL mRNA and protein expression was confirmed by qRT-PCR and Western blotting (Supplementary Fig. S2F). In the Ras activity assay, the Ras-GTP level was dramatically reduced in MLL siRNA knockdown (KD) A549 cells, in accordance with decreased activity of pAKT and pERK1/2 (Fig. 2C). Similar results were obtained in H1299 (Kras WT) cells with ectopic expression of KrasG12V after MLL inhibition by siRNA-KD (Fig. 2D). The transfer of exogenous KrasG12V also upregulated MLL protein expression (Fig. 2D). MLL ablation reduced pAKT and pERK1/2 activation in the lung tumors of mice with ATII cell– and Clara cell–specific KrasG12D mutation (Fig. 2E). Overall, MLL inhibition intrinsically attenuated the activation of the Ras pathway.

Figure 2.

MLL ablation inhibits Kras signaling pathway. A, GSEA of RNA-seq (PRJNA830337) showed that Kras, AKT, AKT/mTOR genes were enriched in MLLΔ/Δ (n = 3) compared with MLLf/f (n = 3) lung tissues. Each of the black bars represents a gene in the pathway. BD, Western blotting was used to detect the indicated proteins in MEF (which were treated with 4-OH-TAM for 3 days; B), A549 (which were treated with siNC and siMLL-2 for 3 days; C), and H1299 (KrasG12V overexpression H1299 cells were treated with siNC and siMLL-2 for 3 days; D). E, Representative images of KSf (n = 9), KMSf (n = 9), KSc (n = 6), and KMSc (n = 8) lung tissue sections stained for pAKT and pERK1/2 by IHC. Scale bars, 100 μm. Right, quantification of pAKT and pERK1/2 IHC staining in the lung tissues sections of KSf, KMSf, KSc, and KMSc mice. F, Western blotting was used to detect the indicated proteins in primary MEF cells. Isolation and culture of primary MEF cells as previous methods, and then the cells were treated with 1 μmol/L 4-OH-TAM for 3 days to generate MLLf/f, MLLΔ/Δ, WT, LK, and LKM cells. G, Representative IHC staining for pChk1 in the lung tissues sections of KSf (n = 13), KMSf (n = 9), KSc (n = 6), and KMSc (n = 10) mice. Scale bars, 100 μm. Right, quantification of the pChk1 IHC staining. H, Growth curve of primary WT, LK, and LKM cells during serial passages. n = 3. Data are represented as mean ± SEM in E and G. The P values were determined by two-tailed unpaired t tests.

Figure 2.

MLL ablation inhibits Kras signaling pathway. A, GSEA of RNA-seq (PRJNA830337) showed that Kras, AKT, AKT/mTOR genes were enriched in MLLΔ/Δ (n = 3) compared with MLLf/f (n = 3) lung tissues. Each of the black bars represents a gene in the pathway. BD, Western blotting was used to detect the indicated proteins in MEF (which were treated with 4-OH-TAM for 3 days; B), A549 (which were treated with siNC and siMLL-2 for 3 days; C), and H1299 (KrasG12V overexpression H1299 cells were treated with siNC and siMLL-2 for 3 days; D). E, Representative images of KSf (n = 9), KMSf (n = 9), KSc (n = 6), and KMSc (n = 8) lung tissue sections stained for pAKT and pERK1/2 by IHC. Scale bars, 100 μm. Right, quantification of pAKT and pERK1/2 IHC staining in the lung tissues sections of KSf, KMSf, KSc, and KMSc mice. F, Western blotting was used to detect the indicated proteins in primary MEF cells. Isolation and culture of primary MEF cells as previous methods, and then the cells were treated with 1 μmol/L 4-OH-TAM for 3 days to generate MLLf/f, MLLΔ/Δ, WT, LK, and LKM cells. G, Representative IHC staining for pChk1 in the lung tissues sections of KSf (n = 13), KMSf (n = 9), KSc (n = 6), and KMSc (n = 10) mice. Scale bars, 100 μm. Right, quantification of the pChk1 IHC staining. H, Growth curve of primary WT, LK, and LKM cells during serial passages. n = 3. Data are represented as mean ± SEM in E and G. The P values were determined by two-tailed unpaired t tests.

Close modal

Compared with MLLf/f MEFs, MLL-KO MEFs showed increased cyclin B1 and cyclin D1 expression; however, there was no significant effect on the expression of cyclin A2, CDK2, and CDK4 (Fig. 2F). Interestingly, deletion of MLL distinctly decreased the expression of cell-cycle checkpoint kinase 1 (Chk1) and the levels of phosphorylated Chk1 (pChk1; Fig. 2F). Chk1 is an active transducer kinase at both the S and G2 checkpoints. In the presence of DNA damage, inhibition of Chk1 abrogates G2 arrest, leading to cancer cell death (21). Similar results were obtained in WT, LK, and LKM MEFs: Kras activation markedly promoted cell-cycle regulator expression, and MLL deletion specifically repressed the increases in pChk1 and Chk1 induced by Kras activation (Fig. 2F). Consistently, immunostaining of pChk1 in lung tumor tissues of KSc, KMSc, KSf, and KMSf mice indicated that MLL deletion inhibited its expression (Fig. 2G). Accordingly, we observed a significantly higher proliferative capacity of LK MEFs than WT MEF cells; however, the number of LKM cells declined at passage 5 (Fig. 2H), further indicating that loss of MLL inhibits Kras mutation–induced cell proliferation. We suspect that loss of MLL decreased the activation of Chk1 by Kras, leading to genomic instability and impaired cell survival.

MLL transcriptionally regulates Rasgrf1 expression through H3K4me3

Next, we turned our attention to the impact of MLL regulating on the activation of Ras. The Ras-GEFs family proteins, which is Ras activitors, regulate the cycle of Ras-GDP to Ras-GTP. We found that loss of MLL reduced expression of Ras activitors from RNA-seq (PRJNA830337). We suspected that whether MLL mediates Ras activity through transcriptional regulation of Ras activitors. As expected, the mRNA expression of Rasgrp1, Rasgrf1, Rapgef5, and RGL1 were suppressed in the lung tissues of MLLΔ/Δ mice (Fig. 3A). However, only Rasgrf1 was consistent with our speculation, which was upregulated by KrasG12D activation and suppressed with MLL ablation in the lung tumors of Scgb1a1-Cre mice (Fig. 3B). To further verify whether Rasgrf1 is regulated by MLL, multiple models were developed, and the findings were validated by qRT-PCR. The expression of Rasgrf1 was repressed in siMLL-treated A549 and MLL-KO primary ATII cells (Fig. 3C and D). In primary MEFs, loss of MLL dramatically attenuated the Rasgrf1 mRNA expression induced by KrasG12D/+ (Fig. 3E). Western blotting was used to detect the changes of Rasgrf1 in siMLL-treated A549 and primary MEFs (Supplementary Fig. S3A). Treatment with MI-3, a small-molecule inhibitor that specifically targets H3K4me3 (19), notably decreased Rasgrf1 expression and Ras activity in A549 cells (Fig. 3F; Supplementary Fig. S3B), suggesting that MLL regulates Rasgrf1 potentially through H3K4me3. We further performed ChIP assays to detect whether MLL directly regulates Rasgrf1 transcription through epigenetic mechanisms. The ChIP assays showed that MLL bound to the Rasgrf1 promoter, and MLL KD notably reduced the binding of MLL in the promoter regions of Rasgrf1, especially in PP1 and PP3 (Fig. 3G). Furthermore, abundant H3K4me3 modifications were detected in Rasgrf1 promoter regions, and MLL KD by siRNA reduced the H3K4me3 level (Fig. 3H). Similar results were detected in MEFs (Supplementary Fig. S3C). These results show that MLL transcriptionally regulates Rasgrf1 expression through H3K4me3.

Figure 3.

MLL regulate Rasgrf1-mediated Kras activity through H3K4me3. A, The mRNA expression of Rasgrp1, Rasgrf1, Rasgrf2, Rapgef5, RGL1, and IGF2 measured by qRT-PCR in the lung tissues of MLLf/f (n = 3) and MLLΔ/Δ (n = 5) mice. The MLLf/f and MLLf/f;UBC-cre mice were intraperitoneally injected with 100 mg/kg TAM to generate MLLf/f and MLLΔ/Δmice. B, Analysis of mRNA levels of indicated genes by qRT-PCR in lung tumors dissected from the WT (n = 3), KSc (n = 5), and KMSc (n = 8) mice. CE, The mRNA expression of MLL, Rasgrf1, and IGF2 was measured by qRT-PCR in A549, primary ATII, and MEF cells. n = 3. F, A549 cells were treated with 1 μmol/L MI-3 for 3 days, and Western blotting was used to detect the indicated proteins. G and H, Schematic representation of the Rasgrf1 gene promoter regions and primer pairs used for ChIP assays. ChIP-qPCR was performed with an anti-MLL and H3K4me3 antibody, respectively, on samples from A549 cells (which were treated with siNC or siMLL-2 for 3 days), and IgG served as the negative control. PPs, n = 3. Data are represented as mean ± SD. The P values were determined by two-tailed unpaired t tests.

Figure 3.

MLL regulate Rasgrf1-mediated Kras activity through H3K4me3. A, The mRNA expression of Rasgrp1, Rasgrf1, Rasgrf2, Rapgef5, RGL1, and IGF2 measured by qRT-PCR in the lung tissues of MLLf/f (n = 3) and MLLΔ/Δ (n = 5) mice. The MLLf/f and MLLf/f;UBC-cre mice were intraperitoneally injected with 100 mg/kg TAM to generate MLLf/f and MLLΔ/Δmice. B, Analysis of mRNA levels of indicated genes by qRT-PCR in lung tumors dissected from the WT (n = 3), KSc (n = 5), and KMSc (n = 8) mice. CE, The mRNA expression of MLL, Rasgrf1, and IGF2 was measured by qRT-PCR in A549, primary ATII, and MEF cells. n = 3. F, A549 cells were treated with 1 μmol/L MI-3 for 3 days, and Western blotting was used to detect the indicated proteins. G and H, Schematic representation of the Rasgrf1 gene promoter regions and primer pairs used for ChIP assays. ChIP-qPCR was performed with an anti-MLL and H3K4me3 antibody, respectively, on samples from A549 cells (which were treated with siNC or siMLL-2 for 3 days), and IgG served as the negative control. PPs, n = 3. Data are represented as mean ± SD. The P values were determined by two-tailed unpaired t tests.

Close modal

The Rasgrf1 gene is an imprinted gene; the imprinted Rasgrf1 locus is methylated on the paternal allele within a differentially methylated region (DMR) on the 5′ side of the promoter, and differentially methylated domain methylation is required for imprinted Rasgrf1 expression (22). We found that another classical imprinted gene, IGF2, was also regulated by MLL in the indicated models (Fig. 3AE). We wondered whether MLL regulates Rasgrf1 through DNA methylation. However, the inhibition of MLL did not affect the expression of DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) or the expression of the insulator CTCF (Supplementary Fig. S3D and S3E). The ChIP results indicated that MLL inhibition modestly increased the binding of CTCF with imprinting control regions (Supplementary Fig. S3F). Although the expression of the demethylase TET1 was downregulated by MLL KD (Supplementary Fig. S3G), bisulfite modification assays indicated that MLL deletion did not alter the DNA methylation of the DMRs or promoter regions in the lung tumors of KSc and KMSc mice and primary MEFs (Supplementary Fig. S3H and S3I). This finding further suggested that the regulation of Rasgrf1 transcription by MLL depends on H3K4me3 and not on traditional imprinting regulation.

To further verify the essential role of Rasgrf1 in MLL-regulated Kras activation, two distinct Rasgrf1 siRNAs were transfected into H1299 or H157 cells that had also been transfected with KrasG12V constructs. Reduced Rasgrf1 mRNA was detected by qPCR, and the Ras-GTP levels were significantly reduced in the siRNA treatment groups (Supplementary Fig. S3J). Next, A549 cells were cotreated with siMLL and transfected with a Rasgrf1 construct. Western blotting showed significant inhibition of Ras-GTP activity and its downstream pathways (pAKT and pERK1/2) after siMLL treatment alone; furthermore, restoration of Rasgrf1 expression reactivated Ras activity and its downstream pathways (Supplementary Fig. S3K). On the basis of the above results, Rasgrf1 is necessary for MLL-regulated Kras activity.

MLL inhibition enhances the chemotherapy sensitivity of lung cancer cells

We further determined the biological importance of the MLL-regulated Kras pathway in controlling the lung cancer phenotype. After transfection with siNC or siMLL, A549 cells were treated with the chemotherapeutic agent cisplatin (DDP) or etoposide (ET). Cholecystokinin-8 (CCK8) assay results showed that MLL inhibition significantly increased the sensitivity of A549 cells to DDP or ET (Fig. 4A and B). The colony formation assays also showed that MLL KD significantly increased the DDP and ET sensitivity of A549 cells in a dose-dependent manner (Fig. 4C and D). Next, a variety of lung cancer cells were treated with MI-3, and cell viability was detected by CCK8 assay. The results revealed that A549 (Krasmut) and H1944 (Krasmut) cells were more sensitive than H1299 (KrasWT, P53null), H157 (KrasWT, P53mut), and H1975 (KrasWT, EGFRmut) cells to MI-3 (Supplementary Fig. S4A). In addition, MI-3 was more likely to increase the sensitivity of lung cancer cells containing Kras mutations to DDP (Fig. 4E). It is reported that H1944 cells also contains KrasG13D mutation (23). KrasG13D mutation has elevated intrinsic exchange activity relative to WT Ras, allowing KrasG13D to accumulate in a persistently GTP-bound state. Therefore, we speculate that MI-3 may target lung cancer cells harboring Kras mutations. To prove this hypothesis, a rescue experiment that Kras WT H1299 or H157 cells stably expressing KrasG12V were cotreated with MI-3 and DDP, and Kras mRNA expression was detected by qRT-PCR (Supplementary Fig. S4B). The results showed that MI-3 specifically enhanced the sensitivity of KrasG12V-overexpressing lung cancer cells but not WT lung cancer cells to DDP (Fig. 4F; Supplementary Fig. S4C). These results suggest that inhibition of MLL specifically blocks the survival pathway and that the MLL pathway might serve as a target for chemotherapeutic drugs in Kras-mutant lung cancer cells.

Figure 4.

MLL inhibition enhancing chemotherapy sensitivity of lung cancer cells. A–D, A549 cells were treated with siNC or siMLL-2, and the generated cells were treated with cisplatin or ET. The cell proliferation was measured by the CCK8 assay at day 3 (A and B), and the colony-forming activity was determined at day 7 (C and D). Right, quantification of colony numbers. n = 3. E, The A549, H1299, H157, H1975, and H1944 cells were cotreated with MI-3 and DDP for 72 hours and cell proliferation was measured by CCK8 assays. n = 3. F, The H157 cells were stably transfected with the empty vector or KrasG12V-overexpressing plasmid via the lentiviral. The transfected cells were cotreated with MI-3 and DDP for 72 hours and cell proliferation was measured by CCK8 assays. n = 3. Data are represented as mean ± SD in AF. The P values were determined by two-tailed unpaired t tests.

Figure 4.

MLL inhibition enhancing chemotherapy sensitivity of lung cancer cells. A–D, A549 cells were treated with siNC or siMLL-2, and the generated cells were treated with cisplatin or ET. The cell proliferation was measured by the CCK8 assay at day 3 (A and B), and the colony-forming activity was determined at day 7 (C and D). Right, quantification of colony numbers. n = 3. E, The A549, H1299, H157, H1975, and H1944 cells were cotreated with MI-3 and DDP for 72 hours and cell proliferation was measured by CCK8 assays. n = 3. F, The H157 cells were stably transfected with the empty vector or KrasG12V-overexpressing plasmid via the lentiviral. The transfected cells were cotreated with MI-3 and DDP for 72 hours and cell proliferation was measured by CCK8 assays. n = 3. Data are represented as mean ± SD in AF. The P values were determined by two-tailed unpaired t tests.

Close modal

MI-3 inhibits the growth of Kras-mutated lung cancer cells in vivo

Next, we explored the potential impact of MLL in controlling lung tumor growth in vivo. The A549 lung tumor xenograft model construction and MI-3 administration protocols are described in Supplementary Fig. S5A. A549 tumor growth increased exponentially over time in the control mice, and the MI-3 treatment group showed suppressed tumor growth over the period of treatment in comparison with the controls (Fig. 5A). Mice were euthanized at day 14, and the xenograft tumors were dissected and photographed (Supplementary Fig. S5B). There was no difference in the change in body weight between the two groups, suggesting that MI-3 does not have high toxicity (Supplementary Fig. S5C). Immunostaining of Ki67 and cleaved caspase-3 indicated that MI-3 treatment significantly inhibited tumor cell proliferation and promoted apoptosis (Fig. 5B and C). Consistent with the in vitro results, MI-3 treatment also inhibited the expression of Rasgrf1, pAKT, pERK1/2, and Chk1 (Fig. 5B and C). We further generated a Kras WT H1299 lung tumor xenograft model. We found that MI-3 did not effectively inhibit H1299 cell xenograft growth in vivo (Supplementary Fig. S5D). Interestingly, transfection of a KrasG12V construct rapidly promoted the growth of H1299 cells in vivo, and the same dose of MI-3 effectively inhibited the growth of H1299 tumors transfected with the KrasG12V construct with low toxicity (Supplementary Fig. S5D and S5E). The expression of KrasG12V significantly increased Ki67, Rasgrf1, and pERK1/2 and decreased cleaved caspase-3 staining compared with that seen in WT H1299 cell xenograft tumors (Fig. 5D and E). The reductions in Ki67, Rasgrf1, and pERK1/2 staining and increases in cleaved caspase-3 staining were observed in tumors treated with MI-3 (Fig. 5D and E). These results further demonstrate that MI-3 predominantly inhibits the growth of lung tumors harboring Kras mutations by targeting MLL.

Figure 5.

MI-3 specifically suppresses Kras-mutant lung tumor growth in vivo. A, A total of 3 × 106 A549 cells were transplanted into nude mice, and the tumor volume (mean ± SD) was measured every 4 days at the indicated time points after MI-3 intraperitoneal injection. n = 7 for per group. B, IHC staining for Ki67, cleaved caspase-3, Rasgrf1, pAKT, pERK1/2, and Chk1 in the A549-Con (n = 6) and A549-MI-3 group (n = 7). Scale bars, 100 μm. C, Quantification of IHC staining in indicated xenograft tumor sections. D, The H1299 cells were stably transfected with either empty vector or KrasG12V-overexpressing plasmid via lentiviral, and the generated cells were transplanted into nude mice. After 2 weeks of MI-3 intraperitoneal injection, mice were sacrificed, and tumors were removed for analysis. IHC staining for Ki67, cleaved caspase-3, Rasgrf1, and pERK1/2 in the H1299-vector-Con (n = 5), H1299-vector-MI-3 (n = 10), H1299- KrasG12V-Con (n = 5), and H1299-KrasG12V-MI-3 (n = 10) group. Scale bars, 100 μm. E, Quantification of IHC staining in indicated xenograft tumor sections. Data are represented as mean ± SEM. The P values were determined by two-tailed unpaired t tests.

Figure 5.

MI-3 specifically suppresses Kras-mutant lung tumor growth in vivo. A, A total of 3 × 106 A549 cells were transplanted into nude mice, and the tumor volume (mean ± SD) was measured every 4 days at the indicated time points after MI-3 intraperitoneal injection. n = 7 for per group. B, IHC staining for Ki67, cleaved caspase-3, Rasgrf1, pAKT, pERK1/2, and Chk1 in the A549-Con (n = 6) and A549-MI-3 group (n = 7). Scale bars, 100 μm. C, Quantification of IHC staining in indicated xenograft tumor sections. D, The H1299 cells were stably transfected with either empty vector or KrasG12V-overexpressing plasmid via lentiviral, and the generated cells were transplanted into nude mice. After 2 weeks of MI-3 intraperitoneal injection, mice were sacrificed, and tumors were removed for analysis. IHC staining for Ki67, cleaved caspase-3, Rasgrf1, and pERK1/2 in the H1299-vector-Con (n = 5), H1299-vector-MI-3 (n = 10), H1299- KrasG12V-Con (n = 5), and H1299-KrasG12V-MI-3 (n = 10) group. Scale bars, 100 μm. E, Quantification of IHC staining in indicated xenograft tumor sections. Data are represented as mean ± SEM. The P values were determined by two-tailed unpaired t tests.

Close modal

MLL expression is positively correlated with poor prognosis in NSCLC

Because we observed a crucial role for MLL in controlling LUAD tumorigenesis, we wonder whether MLL expression is altered in patient primary lung tumors. We found that MLL mRNA expression was significantly higher in lung cancer cells than in BEAS-2B normal lung bronchial cells (Supplementary Fig. S6A). Analysis of mRNA expression via TCGA database showed that MLL mRNA expression was significantly higher in LUAD and LIHC tumor tissues than in normal control tissues but not in COAD, PAAD or (PARD (Supplementary Fig. S6B). Furthermore, IHC was performed to detect targets in human NSCLC. We found that compared with normal tissues, lung tumor tissues had higher levels of Kras, MLL, Rasgrf1, Chk1, and pChk1 (Fig. 6A). Subsequently, the correlation analysis showed that MLL was positively correlated with Kras, Rasgrf1, Chk1, and pChk1 expression in 27 NSCLC samples (Fig. 6BE). We also found a positive correlation of MLL with Rasgrf1 and Chk1 in 535 NSCLC samples from TCGA database (Fig. 6F and G). In addition, the statistical analysis showed that 28.6% of samples were moderately differentiated and 71.4% of samples were poorly differentiated in the MLL-high NSCLC group, while 81.8% of samples were moderately differentiated and 18.2% of samples were poorly differentiated in the MLL-low NSCLC group (Fig. 6H). We obtained 183 cases of LUAD from TCGA database for the Kaplan–Meier survival analysis. The result showed that, according to MLL expression level, 2.5-year overall survival was lower for MLL-high patients than for MLL-low patients (Fig. 6I). These results indicate the essential regulatory role of MLL in the activation of the Kras pathway and further suggest that MLL expression is positively correlated with poor prognosis in NSCLC.

Figure 6.

MLL expression is positively correlated with poor prognosis in NSCLC. A, IHC staining for the indicated proteins in clinical NSCLC samples, n = 27. Scale bars, 60 μm. BE, Correlation analysis between MLL and Kras, Rasgrf1, pChk1, and Chk1 expression in NSCLC samples, n = 27. FG, Correlation analysis between mRNA levels of MLL and Rasgrf1 expression, and MLL and Chk1 expression in NSCLC samples in TCGA database. n = 535. The Spearman correlation and P values by Spearman test are indicated in BE and F and G. H, Pie diagrams show the number of samples with the degree of tumor differentiation in MLL-low and MLL-high NSCLC samples, n = 25. High, MLL+ area ≥ 10,000; low, MLL+ area < 10,000. I, Kaplan–Meier survival analysis for LUAD according to MLL expression. A total of 183 patients with LUAD obtained from TCGA were plotted according to the normal distribution of MLL expression, specifying 0.44–1.84 as the MLL-low expression group and 2.317–5.61 as the MLL-high expression group. The HRs and P values by log-rank (Mantel–Cox) test are indicated in figure.

Figure 6.

MLL expression is positively correlated with poor prognosis in NSCLC. A, IHC staining for the indicated proteins in clinical NSCLC samples, n = 27. Scale bars, 60 μm. BE, Correlation analysis between MLL and Kras, Rasgrf1, pChk1, and Chk1 expression in NSCLC samples, n = 27. FG, Correlation analysis between mRNA levels of MLL and Rasgrf1 expression, and MLL and Chk1 expression in NSCLC samples in TCGA database. n = 535. The Spearman correlation and P values by Spearman test are indicated in BE and F and G. H, Pie diagrams show the number of samples with the degree of tumor differentiation in MLL-low and MLL-high NSCLC samples, n = 25. High, MLL+ area ≥ 10,000; low, MLL+ area < 10,000. I, Kaplan–Meier survival analysis for LUAD according to MLL expression. A total of 183 patients with LUAD obtained from TCGA were plotted according to the normal distribution of MLL expression, specifying 0.44–1.84 as the MLL-low expression group and 2.317–5.61 as the MLL-high expression group. The HRs and P values by log-rank (Mantel–Cox) test are indicated in figure.

Close modal

MLL proteins are evolutionarily conserved chromatin-modifying factors originally identified as part of an epigenetic cellular memory system that plays a widespread role in transcriptional activation and have been shown to globally control multiple cellular processes (8). Multiple somatic mutations of MLL have been detected in diverse solid cancers, including lung cancer (24). TCGA database analysis showed that MLL had different point mutation, amplification, and deep deletion frequencies in small cell lung cancer, LUAD, and lung squamous cell carcinoma (Supplementary Fig. S6C). However, the biological function of MLL in lung carcinogenesis is poorly understood. The current study shows that Kras-induced lung carcinogenesis is dependent on MLL-mediated H3K4 methyltransferase activity. We found significantly higher levels of MLL mRNA in tumor tissues than in adjacent normal tissues. MLL regulates Rasgrf1 transcription through H3K4me3, which further promotes the Ras-GDP to Ras-GTP transition. Therefore, MLL deletion leads to downregulation of Rasgrf1 and thus suppresses Kras activation, resulting in the inhibition of the downstream pathway.

Kras is one of the most common mutated oncogenes in human cancers. Mutant Kras continues to be activated even without the activation of growth factors or receptor tyrosine kinases, leading to continued cell proliferation and ultimately cancer (3). Because of Ras lacks classic and tractable drug binding sites, identification and development of drugs that specifically target Ras are challenging. Recently, sotorasib and adagrasib were evaluated in phase I–III trials for the treatment of locally advanced or metastatic NSCLC with KrasG12C mutations, heralding a new era of precision oncology (25). Although the results suggest that sotorasib and adagrasib have a highly selective inhibitory effect on cancer cells containing the KrasG12C mutation (26), the use of most targeted drugs eventually results in drug resistance, and sotorasib and adagrasib are no exception (27). Novel Kras modifications, including amplification of the Kras gene, secondary mutation of KrasG12C, and activation of WT Kras, have been found to be the main mechanisms of drug resistance (27). MI-3 and its derivatives may be potential therapeutic agents for the treatment of leukemias induced by MLL fusion proteins (19). In addition, the MLL complex works as a crucial coactivator of androgen receptor (AR) signaling, and targeted inhibition of menin/MLL interactions by MI-136 or MI-503 can effectively block AR signaling and inhibit the growth of castration-resistant tumors in vivo, indicating that the MLL complex is a potential therapeutic target in advanced prostate cancer (28). Importantly, we found that MI-3 specifically inhibits Kras-mutant lung cancer cells. MLL activates the activity of mutant Kras by promoting Rasgrf1 expression; on the other hand, activation of this Kras mutant promotes MLL expression through an unknown mechanism (Supplementary Fig. S6D and S6E), forming an interesting positive feedback loop that promotes the malignant transformation of lung epithelial cells. MI-3 effectively disrupts the positive feedback loop between Kras and MLL. Our results showed that MI-3 specifically inhibited the growth of Kras-mutated A549 cells in vivo while having no or minimal effect on Kras WT lung cancer cells. In summary, specific blockade of the MLL-Rasgrf1 pathway may be a potential targeted therapeutic strategy for the treatment of tumors that contain Kras mutations. These results provide helpful ideas for the further design of treatments for Kras-mutant malignancies using MLL-specific inhibitors.

Although the tumor incidence was decreased in mice with ATII-cell-specific KrasG12D/+ mutation and MLL ablation, the mortality and survival of these mice did not differ from those of the KSf group. Pathologic analysis showed severe alveolar destruction with positive PAS staining and negative Alcian blue staining, features similar to those seen in the rare lung disease PAP (29). We also found that the alveolar surfactant protein sftpa/b/d was significantly increased in MLL KO lung tissues in the RNA-seq data analysis (Supplementary Fig. S6F). Pulmonary surfactant reduces the tension in the alveolar fluid airplane and prevents lung collapse at the end of expiration. Therefore, we suspect that MLL deficiency leads to impaired survival of ATII alveolar epithelial cells and secretion of pulmonary surfactant and eventually causes abnormal alveolar function and structural damage, but the mechanism needs to be further elucidated.

In conclusion, WT MLL is critical for the activation of the Kras mutant and its induction of LUAD, and specifically targeting MLL can effectively inhibit Kras-mutant lung cancer. The current study further demonstrated an interesting combined effect of genetic and epigenetic mechanisms on lung tumorigenesis.

No disclosures were reported.

L.-Y. Zhu: Resources, data curation, formal analysis, investigation, methodology, writing–original draft. J.-B. Yuan: Formal analysis, investigation, methodology. L. Zhang: Data curation, formal analysis, investigation. C.-X. He: Investigation. X. Lin: Investigation. B. Xu: Writing–review and editing. G.-H. Jin: Conceptualization, formal analysis, supervision, funding acquisition, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing.

This work was supported by grants from the National Natural Science Foundation of China 82073118 and U1605224 (to G.-H. Jin), 81672793 (to B. Xu.)

The authors thank Dr. Patricia Ernst for providing the MLLf/f mice.

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

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Supplementary data