Patent Application
20250075210
The invention relates to cancer treatment that targets YTHDF1-ARHGEF2 axis and includes compositions that targeting the same.
Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths worldwide.1 CRC incidence and death rates have been increasing in younger adults.1 Metastasis is the major cause of cancer death. In 50-60% of CRC patients, CRC eventually develops metastatic disease, most of which are unresectable liver and lung metastases.2,3 Metastatic CRC are treated with chemotherapy, targeted therapy and immunotherapy.2 Chemotherapy and targeted therapy have demonstrated limited efficacy in the control of CRC metastasis. Metastatic CRCs with microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) benefit from anti-PD1/PD-L1 immunotherapy. However, it is ineffective in the vast majority of CRCs that are microsatellite stable or mismatch repair-proficient (MSS/pMMR). Hence, there is an urgent need to identify novel druggable targets and prediction markers for CRC.
Over the past decade, intensive efforts such as The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) have led to the comprehensive genetic, epigenetic and transcriptomic characterization of CRC.4,5 In contrast to the well-established roles of these molecular determinants, post-transcriptional mechanisms that contribute to tumorigenesis remain poorly understood. Epitranscriptomics, which refers to post-transcriptional modifications of RNA transcriptome, plays critical regulatory roles in gene expression.6 Recent advances in sequencing technologies enabled examination of RNA modifications on a genome-wide scale,7,8 revealing significant dysregulation of RNA modifications during tumorigenesis.9
RNA N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic mRNAs.10 m6A is dynamically regulated by the action of m6A writers (METTL3/METTL14/WTAP protein complex) and m6A erasers (FTO and ALKBH5). m6A modification in turn mediates the recruitment of m6A readers that associate m6A-modified RNAs to mRNA processing enzymes, influencing RNA export, splicing, translation and degradation.10 Three classes of m6A readers have been characterized, based on the manner by which they bind to m6A modified RNAs. 11 One major class is the YT521-B homology (YTH) domain family of proteins that directly bind to m6A modified sites (YTHDF1/2/3 and YTHDFC1/2).10 YTHDF1 has been identified as translation-facilitating m6A reader that recruits translation machinery to its target mRNAs in the cytoplasm.12 Recent studies revealed that YTHDF1 plays crucial roles in various physiological processes such as learning and memory, CD8+ T cell priming and axon guidance.13-15 However, it remains to reveal whether and how m6A modifications translate pro-tumorigenic signals via YTHDF1.
Applicant sought to address the consequence of m6A modifications in CRC mediated by YTHDF1. Applicant demonstrated that YTHDF1 is overexpressed in CRC, and its expression is associated with CRC metastasis. Integrative multiomic analysis highlighted a novel oncogenic epitranscriptome axis YTHDF1-m6A-ARHGEF2. The tumorigenic functions of this axis were validated in CRC cell lines, 3D organoid culture and Ythdf1 transgenic mice. Moreover, ARHGEF2 siRNA drug encapsulated by lipid nanoparticles (LNP) was developed for in vivo tumor treatment. Taken together, our work suggests YTHDF1-m6A-ARHGEF2 as a novel therapeutic target for CRC.
Accordingly, in an aspect, there is provided a method for the treatment of cancer in a subject in need thereof, the method comprising downregulating YTHDF1 or ARHGEF2.
In a further aspect, there is provided a nucleic acid molecule capable of selectively inhibiting, at least partially, YTHDF1 or ARHGEF2 expression.
In a further aspect, there is provided a pharmaceutical composition comprising the nucleic acid molecule described herein along with a pharmaceutically acceptable carrier.
In a further aspect, there is provided a use of the nucleic acid molecule described herein, in the preparation of a medicament for the treatment of cancer in a subject in need thereof.
These and other features of the preferred embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:
In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it is understood that the invention may be practiced without these specific details.
N6-methyladenosine (m6A) governs the fate of RNAs through m6A readers. Colorectal cancer (CRC) exhibits aberrant m6A modifications and expression of m6A regulators. However, how m6A readers interpret oncogenic m6A methylome to promote malignant transformation remains to be illustrated.
Ythdf1 knockout mouse was generated to determine the effect of Ythdf1 in CRC tumorigenesis in vivo. Multiomic analysis of RNA-sequencing, m6A methylated RNA immunoprecipitation sequencing, YTHDF1 RNA immunoprecipitation sequencing and proteomics were performed to unravel targets of YTHDF1 in CRC. The therapeutic potential of targeting YTHDF1-m6A-ARHGEF2 was evaluated using siRNA encapsulated by lipid nanoparticles (LNP).
DNA copy number gain of YTHDF1 is a frequent event in CRC and contributes to its overexpression. High expression of YTHDF1 is significantly associated with metastatic gene signature in patient tumors. Ythdf1 knockout in mice dampened tumor growth in an inflammatory CRC model. YTHDF1 promotes cell growth in CRC cell lines and primary organoids, and lung and liver metastasis in vivo. Integrative multiomics analysis identified RhoA activator ARHGEF2 as a key downstream target of YTHDF1. YTHDF1 binds to m6A sites of ARHGEF2 mRNA, resulting in enhanced translation of ARHGEF2. Ectopic expression of ARHGEF2 restored impaired RhoA signaling, cell growth and metastatic ability both in vitro and in vivo caused by YTHDF1 loss, verifying that ARHGEF2 is a key target of YTHDF1. Finally, ARHGEF2 siRNA delivered by LNP significantly suppressed tumor growth and metastasis in vivo.
We identify a novel oncogenic epitranscriptome axis of YTHDF1-m6A-ARHGEF2, which regulates CRC tumorigenesis and metastasis. siRNA-delivering LNP drug validated the therapeutic potential of targeting this axis in CRC.
In an aspect, there is provided a method for the treatment of cancer in a subject in need thereof, the method comprising downregulating YTHDF1 or ARHGEF2.
The methods, uses, and compositions described herein include embodiments relating to agents capable of inhibiting, downregulating, or abolishing the activity and/or the expression of YTHDF1 or ARHGEF2, total or partially, or any combination of one or more such inhibitor agents. As long as the agent possesses the inhibitory function (e.g., inhibits YTHDF1 or ARHGEF2 expression and/or activity), the inhibitor agent may be selected from any class of compound. Thus, the inhibitors as used herein refer to any compound that reduces, inhibits, downregulates, or abolishes the expression and/or function of YTHDF1 or ARHGEF2, total or partially, or an agent suitable for neutralizing, reducing, or inhibiting the expression or function of YTHDF1 or ARHGEF2. The inhibitors described herein may exert action by any mechanism including, for example, by binding to YTHDF1 or ARHGEF2 transcripts.
In some embodiments, the method comprises downregulating the YTHDF1-ARHGEF2 axis.
In some embodiments, downregulating ARHGEF2 comprises administration of a nucleic acid molecule to the subject, the nucleic acid molecule capable of selectively inhibiting, at least partially, YTHDF1 or ARHGEF2 expression.
Examples of nucleic acid molecules include antisense oligonucleotides and in addition, those capable of mediating RNA interference which include a duplex RNA such as an SiRNA (small interfering RNA), miRNA (micro RNA), shRNA (short hairpin RNA), ddRNA (DNA-directed RNA), piRNA (Piwi-interacting RNA), or rasiRNA (repeat associated siRNA), and modified forms thereof.
In some embodiments, the nucleic acid molecule is a shRNA, siRNA, mRNA or antisense oligonucleotide targeted to ARHGEF2 or YTHDF1.
In some embodiments, the siRNA comprises sense strand GGAUCUACCUGUCACUACUtt (SEQ ID NO. 1) and antisense sense strand AGUAGUGACAGGUAGAUCCag (SEQ ID NO. 2).
In some embodiments, the shRNA comprises shYTHDF1-1: 5′-CCCAGATGGATCTGCATTTAT-3′ (SEQ ID NO. 3); shYTHDF1-2: 5′-CGACATCCACCGCTCCATTAA-3′ (SEQ ID NO. 4); shARHGEF2-1: 5′-GTGCTATGCCTGTAACAAG-3′ (SEQ ID NO. 5); or shARHGEF2-2: 5′-GACGAAGCAGAGGTAATCT-3′ (SEQ ID NO. 6).
The nucleic acid molecules and methods of this invention may be pooled, or used in combination to down regulate the expression of genes that encode ARHGEF2 or YTHDF1.
The term “oligonucleotide” as used herein refers to a nucleic acid molecule comprising from about 1 to about 100 nucleotides, more preferably from 1 to 80 nucleotides, and even more preferably from about 4 to about 35 nucleotides. This may include nucleic acid molecules of variable length that correspond either to the sense strand or to the non-coding strand of a target nucleic acid sequence.
“Antisense oligonucleotides” are complementary to a region of a target gene and are capable of hybridizing to the target gene sequence and inhibiting gene expression. Gene expression is inhibited through hybridization of an AON to a specific messenger RNA (mRNA) sense target according to the Watson-Crick base pairing, typically in which adenosine and thymidine (uracil in mRNA) or guanosine and cytidine interact through hydrogen bonding. Without being bound to any theory, two mechanisms are generally thought to account for these effects, the first being hybridization with impaired translation of targeted mRNA, the second being the induction of RNase H or similar enzymes with associated degradation of target mRNA.
Oligonucleotide compounds in accordance with the present invention also include siRNAs (small interfering RNAs) and the RISCs (RNA-induced silencing complexes) containing them that result from the RNAi (RNA interference) approach. The RNAi approach is a tool for the inhibition of target gene expression. RNAi is based on an ancient anti-viral defence mechanism in lower eukaryotes. It is induced by double-stranded RNA and its processing to typically 21-23 nt siRNAs, which cause the degradation of homologous endogenous mRNA after hybridizing to the target mRNA in a single stranded fashion with the assistance of the RISC complex. The way in which RNAi inhibits target gene expression remains to be fully elucidated, but presently, RNAi serves as an attractive choice approach to generate loss-of-function phenotypes across a broad spectrum of eukaryotic species, such as nematodes, flies, plants, fungi and mammals.
A short hairpin RNA or small hairpin RNA (shRNA/Hairpin Vector) is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors.
Oligonucleotide compounds in accordance with the present invention also include microRNA (miRNA). “MicroRNA” are single-stranded RNA molecules, typically of about 21-23 nucleotides in length, which regulate gene expression in a hybridization dependent manner. Typically, miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, typically at the 3′end of the mRNA, and their main function is to downregulate gene expression.
In some embodiments, the nucleic acid molecule is administered to the subject in a lipid nanoparticle as delivery vehicle.
The composition and methods disclosed herein can also be used in treating various kinds of malignant tumors in a subject.
In some embodiments, the cancer is selected from the group consisting of colorectal adenocarcinoma, stomach adenocarcinoma, lung adenocarcinoma, breast carcinoma, cholangiocarcinoma, liver hepatocellular carcinoma, head/neck squamous cell carcinoma, uterine corpus endometrial carcinoma, high-risk Wilms tumor, esophageal carcinoma, bladder urothelial carcinoma, kidney renal papillary cell carcinoma, prostate adenocarcinoma, giolblastoma multiforme, cervical squamous cell carcinoma/endocervical adenocarcinoma, pheochromocytoma/paraganglioma, and pancreatic adenocarcinoma. Preferably, the cancer is colorectal adenocarcinoma.
In an aspect, there is provided a nucleic acid molecule capable of selectively inhibiting, at least partially, YTHDF1 or ARHGEF2 expression.
In some embodiments, the nucleic acid molecule is a siRNA. In other embodiments, the nucleic acid molecule is a miRNA or an antisense oligonucleotide.
In some embodiments, the nucleic acid molecule is encapsulated within a lipid nanoparticle.
In some embodiments, the nucleic acid molecule described herein is for use in the treatment of cancer in a subject in need thereof.
In an aspect, there is provided a use of the nucleic acid molecule described herein, in the preparation of a medicament for the treatment of cancer in a subject in need thereof.
In a further aspect, there is provided a pharmaceutical composition comprising the nucleic acid molecule described herein along with a pharmaceutically acceptable carrier.
As used herein, “pharmaceutically acceptable carrier” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent.
As used herein, “therapeutically effective amount” refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects.
Abbreviations used herein include the following: 2′-OMe, 2′-O-Methyl; AOM, azoxymethane; BP, Biological Process; CRC, colorectal cancer; CRISPR, clustered regularly interspaced short palindromic repeats; CPTAC, Clinical Proteomic Tumor Analysis Consortium; DSS, dextran sulfate sodium; FFPE, formalin-fixed paraffin embedded; FDA, Food and Drug Administration; GSEA, Gene Set Enrichment Analysis; GO, Gene Ontology; GEF, guanine nucleotide exchange factor; IHC, immunohistochemistry; IF, immunofluorescence; iTRAQ, isobaric tagging for relative and absolute quantification; KEGG, Kyoto Encyclopedia of Genes and Genomes; LMB, Leptomycin B; LNP, lipid nanoparticles; m6A, N6-methyladenosine; MeRIP, methylated RNA immunoprecipitation; mRNA, messenger RNA; MSI, microsatellite instability; NMD, nonsense-mediated mRNA decay; PCR; quantitative polymerase chain reaction; PmR, protein-to-mRNA; PDI, Polydispersity Index; qPCR, quantitative PCR; Ribo-seq, ribosome profiling; RNA-seq, RNA-sequencing; RIP, RNA immunoprecipitation; ROCK, Rho-associated protein kinase; sgRNA, single-guide RNA; shRNA, short hairpin RNA; TCGA, The Cancer Genome Atlas; UTR, untranslated region; and WT, wildtype.
The advantages of the present invention are further illustrated by the following examples. The examples and their particular details set forth herein are presented for illustration only and should not be construed as a limitation on the claims of the present invention.
151 snap-frozen paired CRC tumors and adjacent non-tumor tissues for quantitative PCR (qPCR) analysis, 12 snap-frozen paired CRC tumors and adjacent non-tumor tissues for Western blot analysis and 22 formalin-fixed paraffin embedded (FFPE) tissue blocks from matched CRC and non-tumor mucosa were obtained from CRC patients who underwent surgery at Prince of Wales Hospital, The Chinese University of Hong Kong.16 Biopsy samples from primary CRC tumor and adjacent non-tumor were obtained from CRC patients at the time of operation before any therapeutic intervention. 31 FFPE tissue blocks from matched CRC and non-tumor mucosa were kindly provided by Dr. Xiaohong Wang from Peking University Cancer Hospital. Tissue microarray slides containing 208 CRC cases were kindly provided by Dr. Wei Kang from Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong. The study protocols have been approved by the Clinical Research Ethics Committee of Prince of Wales Hospital, The Chinese University of Hong Kong and Peking University Cancer Hospital. All patients provided written informed consent for obtaining the study specimens. This study was carried out in accordance with the Declaration of Helsinki of the World Medical Association.
The CRC cell lines HCT116 (Cat. #CCL-247), HT-29 (Cat. #HTB-38), RKO (Cat. #CRL-2577), DLD-1 (Cat. #CCL-221), LoVo (Cat. #CCL-229), LS 180 (Cat. #CL-187) and SW1116 (Cat. #CCL-233) were obtained from the American Type Culture Collection (ATCC). Cell lines were maintained according to protocols from ATCC. Human colon cancer patient-derived organoids PDO828 and PDO816 were kindly provided by Dr. Catherine Adell O'Brien in Princess Margaret Cancer Center in the University of Toronto. Organoids were cultured in Corning Growth Factor Reduced Matrigel matrix (Corning) in advanced DMEM/F12 medium (Thermo Fisher Scientific, Cat. #12634028) supplemented with 1% Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. #15140148), HEPES (10 μM; Thermo Fisher Scientific), GlutaMAX™ Supplement (1:100; Thermo Fisher Scientific), serum free B-27™ Supplement (1:50; Thermo Fisher Scientific), 1.25 mM N-Acetyl-L-cysteine (MilliporeSigma), 10 nM [Leu15]-Gastrin I human (MilliporeSigma), 50 ng/mL recombinant murine epithelial growth factor (Thermo Fisher Scientific), 100 ng/mL murine Noggin (Peprotech) and 0.5 μM A 83-01 (Tocris Bioscience).m1
Plasmids and siRNAs
The full-length Flag-tagged YTHDF1 cDNA (NM_017798.4) was amplified and cloned into the pCDNA3.1+ expression vector (Thermo Fisher Scientific) and lentiviral vector pLVX-Puro (Takara Bio USA). The full-length Flag-tagged ARHGEF2 cDNA (NM 001162383.2) was amplified and cloned into the pCDNA3.1+ expression vector (Thermo Fisher Scientific) and lentiviral vector pLV-Neo (Inovogen Tech. Co.). pCDNA3.1-Flag-DACT2 was constructed as described previouslym2. YTHDF1, ARHGEF2 and negative control (shCtrl) shRNAs were cloned into lentivirus shRNA expression plasmid pLVshRNA-puro (Inovogen Tech. Co.). The shRNA target sequences were as below: shCtrl: 5′-CCACATGAAGCAGCACGACTT-3′; shYTHDF1-1: 5′-CCCAGATGGATCTGCATTTAT-3′; shYTHDF1-2: 5′-CGACATCCACCGCTCCATTAA-3′; shARHGEF2-1: 5′-GTGCTATGCCTGTAACAAG-3′; shARHGEF2-2: 5′-GACGAAGCAGAGGTAATCT-3′). For siRNA knockdown, cells were transfected with YTHDF1 siRNAs (Silencer® Select; Thermo Fisher Scientific) (SiYTHDF1-1: Cat. #4392420-s29743; SiYTHDF1-2: Cat. #4392420-s29745), ARHGEF2 siRNAs (Silencer® Select; Thermo Fisher Scientific) (siARHGEF2-1: Cat. #4392420-s17545; siARHGEF2-2: Cat. #4392420-s17546) or control siRNA (siCtrl; Silencer™ Negative Control No. 1 siRNA; Cat. #AM4635; Thermo Fisher Scientific).
Ythdf1 knockout mice were generated using the CRISPR/Cas9 system. Cas9 mRNA and two sgRNAs (sg1 and sg2) were microinjected into fertilized embryos of C57BL/6J mice. Deletion in exon 4 was confirmed by Sanger sequencing. Genotyping was performed by PCR of tail-snip DNA using genotyping primers (data not shown). All mice were housed in a Specific Pathogen Free (SPF) environment for the duration of the study. All experiments in this study were approved by the Institutional Animal Care and Use Committee at Xiamen University.
Cells were fixed with 4% paraformaldehyde/PBS and permeabilized. Nonspecific binding sites were blocked with 1% BSA/PBS. Primary antibodies were applied to the cells at room temperature for 2 h. After PBS wash, secondary antibodies with desired fluorescence probes were applied. Then cells were washed and mounted with ProLong™ Gold Antifade Mountant with DAPI (4′,6-diamidino-2-phenylindole) (Thermo Fisher Scientific). To inhibit nuclear export, 24 h post transfection, Leptomycin B (LMB; MilliporeSigma; dissolved in methanol) was added to the culture medium to a final concentration of 50 nM for 4 h. To stain stress fibre, cells were fixed 16 h after seeding and stained with Alexa Fluor Plus 555 phalloidin solution (Cat. #A30106; Thermo Fisher Scientific; dissolved in DMSO) for 30 min according to manufacturer's instructions.
The CellTiter-Blue Cell Viability Assay kit (Promega) was used to quantify the growth of organoids. For other CRC cells, the growth curves were evaluated by conventional hemocytometer counting chambers.
˜5000 YTHDF1-knockdown,-overexpressing and the corresponding control HCT116 and HT-29 cells were seeded in 96-well plates. Three days after seeding, cell proliferation was assessed by BrdU Cell Proliferation Assay Kit (Cat. #6813; Cell Signaling Technology) following the manufacturer's instructions.
Corning® BioCoat® Growth Factor Reduced Matrigel Invasion Chamber with 8.0 μm PET Membrane (Cat. #354483, Corning) and Corning® 6.5 mm Transwell® with 8.0 μm Pore Polycarbonate Membrane Insert, Sterile (Cat. #3422, Corning) were used to evaluate cell invasion and migration, respectively. CRC cells (YTHDF1 overexpression: 2×104 cells per insert; other assays: 4×104 cells per insert) were suspended in the culture medium containing 1% FBS and seeded into the upper chambers (invasion: Matrigel-coated; migration: uncoated). The culture medium containing 10% FBS was added into the lower chambers as a chemoattractant. After 24˜60 h incubation, cells on the lower surface were fixed and stained for counting.
For xenograft assay, ˜1×106 HCT116 cells were subcutaneously injected into the right flanks of 4˜6 weeks old male nude or NOD/SCID mice. Body weight and tumor volume were measured every 2˜3 days until the endpoint.
The mouse model of lung metastasis was established by tail-vein injection of HCT116 cells into the nude mouse or NOD/SCID mouse. Specifically, HCT116 cells (overexpression: 5×105 cells per mouse; knockdown: 1×106 cells per mouse) were injected intravenously through the tail vein into each 4-6 weeks old male mouse. The health status of the mice was monitored at least once a week at the beginning and daily when the humane intervention points were approaching. The humane intervention points were that the mice in one of the experimental groups were in poor health conditions, such as suppressed activity, being unresponsive to touch, marked hunched posture, dehydration, rough hair coat, dyspnea and weight loss over 20% of initial body weight. The nude mice and NOD/SCID mice were sacrificed 7˜8 and 5˜6 weeks after tail-vein injection, respectively. The lungs from each mouse were excised and embedded in paraffin. The lung metastasis was assessed either by the number of visible pulmonary metastatic nodules or the proportion of tumor metastases in the mouse lung when tumor nodules were indistinguishable.
The mouse model of liver metastasis was established by either tail-vein injection of HCT116 or RKO cells into the NOD/SCID mouse (overexpression: 5×105 cells per mouse; knockdown: 1×106 cells per mouse) or intrasplenic injection of HCT116 cells into the nude mouse or NOD/SCID mouse. In the intrasplenic injection model, enrofloxacin in the drinking water as a prophylactic oral antibiotic was administered to mice 72 h prior to surgery. Mice were anesthetized with Buprenorphine Sustained-Release (SR). ˜1 cm incision was made in the left upper abdominal wall and ˜1 cm incision was made in the peritoneum to expose the mouse spleen. Moistened sterile cotton swab was used to gently exteriorize the spleen. HCT116 cells (overexpression: 5×105 cells per mouse; nanoparticle siRNA drug treatment: 0.75×106 cells per mouse) were injected into each mouse with a 27 G needle. After the spleen was returned to the abdominal cavity, the muscle layer and skin were closed and subcutaneous fluid therapy was administered. The health status, abdominal distension and the size of tumor formed in the spleen were strictly monitored. The humane intervention points were that the mice in one of the experimental groups had marked abdominal distension, marked tumor in the spleen or in poor health conditions. The mice were sacrificed 3˜4 weeks after intrasplenic injection. The mouse liver was excised and fixed for histological examination. The liver metastasis was assessed either by the number of visible liver metastatic nodules or the liver weight and the proportion of tumor metastases in the mouse liver when tumor nodules were indistinguishable. All animal experiments were approved by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong and The University Health Network.
Paraffin sections at 4 μm thickness were dried in a 60° C. oven for 2 h before staining. IHC was performed according to the manufacturer's guidelines using BenchMark XT Automated Slide Staining System (Ventana Medical Systems) with standard antigen retrieval (CC1, pH 8.0, Cat. #950-124, Ventana Medical Systems). The dilutions for YTHDF1 (Cat. #17479-1-AP, Proteintech) were 1:500 for human clinical samples, 1:500 for YTHDF1 knockdown assays and 1:600 for YTHDF1 overexpression assays in mouse metastases models, respectively. The dilution for ARHGEF2 (Cat. #ab155785, Abcam) was 1:500 for all samples. The dilutions for Ki-67 (Cat. #MA5-14520, Thermo Fisher Scientific) were 1:600 for YTHDF1 overexpression assay in mouse liver metastasis model and 1:500 for the rest of the samples. The primary antibodies were incubated for 32 min. Biotinylated anti-rabbit IgG antibody (Cat. #BA-1000-1.5, Vector Laboratories) was added to slides at 1:200 for 12 min. The primary-secondary complex was then visualized with Ventana iVIEW™ DAB Detection Kit (Cat. #760-091, Ventana Medical Systems). The slides were counterstained with Harris hematoxylin, dehydrated in graded alcohol, cleared in xylene and coverslipped in Permount. Sections were evaluated by two pathologists. The expression levels of YTHDF1 and ARHGEF2 were evaluated by the Allred scoring system. For the samples from mouse metastasis models, Allred score in each specific metastatic site from a given sample was evaluated (proportion score+intensity score). Average Allred score for a given sample from mouse metastasis models=sum of all (Allred score×area proportion (%)) in all metastatic sites). For the rest of samples, Allred score=proportion score+intensity score. For the tissue array, CRC tumor samples were categorized into three groups according to Allred score (Group L: Allred score 1˜5, low expression; Group M: Allred score 6, moderate expression; Group H: Allred score 7˜8, high expression).
Total RNAs were purified with RNeasy Mini Kit (Cat. #74106; QIAGEN), and the DNA was digested by RNase-Free DNase Set (Cat. #79254; QIAGEN). RNA-seq libraries were constructed with TruSeq Stranded mRNA Library Prep kit (Cat. #20020595; Illumina) using 4 μg total RNA according to the manufacturer's protocol. Libraries were sequenced as 45 bp single-end reads in duplicates at ˜40 million reads per library using Illumina NextSeq 500 platform (Illumina).
m6A MeRIP-Seq and m6A MeRIP PCR
m6A MeRIP was performed according to the method we developed previously described.m3 Briefly, total RNA from cells in culture was extracted by TRIzol reagent (Cat. #15596018, Thermo Fisher Scientific) and treated with DNase I (Cat. #04716728001, Roche Diagnostics) according to the manufacturer's instructions. 9 ng of E. coli K-12 (Cat. #EC1, MilliporeSigma) total RNA was added to 4 μg of human total RNA sample before RNA fragmentation. For m6A MeRIP, 30 μl of protein A magnetic beads (Cat. #10002D, Thermo Fisher Scientific) and 30 μl of protein G magnetic beads (Cat. #10004D; Thermo Fisher Scientific) were washed by IP buffer (150 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630), resuspended in 500 μl of IP buffer containing 5 μg anti-m6A antibody (Cat. #ABE572, MilliporeSigma) at 4° C. overnight. The antibody-bead mixture was washed and resuspended in 500 μl of the IP reaction mixture containing 4 μg fragmented total RNA, 100 ul of 5×IP buffer, and 5 μl of RNasin Plus RNase Inhibitor (Cat. #N2611, Promega) and incubated for 2 hours at 4° C. The RNA reaction mixture was then washed using a low/high salt-washing method. After washing, the m6A-enriched fragmented RNA was purified using RNeasy Mini Kit (Cat. #74106, QIAGEN). m6A MeRIP INPUT and IP were subjected to library construction using SMARTer Stranded Total RNA-Seq Kit version 2-Pico Input Mammalian (Cat. #634413, Takara/Clontech) according to the manufacturer's protocol. A purified library mix was subjected for 45 bp single-end sequencing at ˜30 million reads per library using a NextSeq 500/550 High Output Kit v2.5 (75 Cycles) (Cat. #20024906, Illumina). m6A levels of the genes were determined by m6A MeRIP qPCR by assessing the percentage of a target gene in IP fraction relative to that in INPUT fraction: % INPUT=2{circumflex over ( )}(Ct of target gene in IP-adjusted Ct of target gene in INPUT).
Briefly, the protein was extracted using a lysis buffer (4% SDS, 20 mM HEPES and 1×protease inhibitor cocktail). Protein concentrations were measured by Bio-Rad protein assay kit (Bio-Rad). 100 μg of protein was tryptic-digested to peptides following filter-aided sample preparation (FASP) protocol in a 30 KD filter unit (MilliporeSigma). The resulting peptides were collected and desalted by C18 ZipTip (MilliporeSigma). 50 μg of protein extract were labeled with iTRAQ® Reagent-8PLEX Multiplex Kit (MilliporeSigma) according to the manufacturer's protocols. Peptides were directly loaded on a self-pack C18 analytical reverse phase column (ID 75 μm×15 cm, 200 Å, 3 μm particles) at a flow rate of 300 nL/min and eluted by a 60 minu LC gradient of 8% to 22% acetonitrile (ACN) in 0.1% formic acid (FA). The eluted peptides were analyzed by Thermo Scientific Orbitrap Fusion Lumos Tribrid Mass Spectrometer machine in a data-dependent acquisition mode, setting as following: 1 microscan for MS1 scans at 120,000 resolution (FWHM at m/z 400), MS2 at 30,000 resolution (FWHM at m/z 400); Full MS mass range: m/z 300-2000; MS/MS mass range: m/z 100-2000. AGC target for MS2 is 50000, maximum injection time is 60 ms, HCD collision energy 35%. The MS raw data is searched against the UniProt Homo sapiens database by software Mascot Daemon (v2.5.1). Search parameters were set as following: trypsin as digestion enzyme and maximum miss cleavage is 2, Precursor Mass Tolerance 20 ppm, Fragment Mass Tolerance 0.02 Da, Carbamidomethyl on Cysteine as fix modification, FDR is set at 0.01. Three biological replicates were included for each group to increase the reliability of isobaric-tags for quantitation.
2×107 HCT116 cells were harvested after 24 h transfection of Flag-tagged YTHDF1 plasmid. The protein was extracted with 1000 μl of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% IGEPAL CA-630, 0.5 mM DTT, 10 μl of RNasin Plus RNase Inhibitor per 1000 μl lysis buffer, 1× protease inhibitor cocktail). 50 μl cell lysate was saved as INPUT, mixed with 1 ml TRIzol. 30 μl of protein A magnetic beads and 30 μl of protein G magnetic beads were washed by NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% IGEPAL CA-630, 0.5 mM DTT), resuspended in 500 μl of NT2 buffer containing 5 μg anti-Flag antibody (Cat. #F1804, MilliporeSigma) at 4° C. overnight. The antibody-bead mixture was washed and resuspended in 995 μl of cell lysate and incubated for 4 h at 4° C. The beads were then washed 8 times with 1 ml ice-cold NT2 buffer and re-suspended with 1 ml TRIzol as IP. RNAs from INPUT and IP were extracted and subjected to library construction using SMARTer Stranded Total RNA-Seq Kit version 2-Pico Input Mammalian (Takara/Clontech) according to the manufacturer's protocol. Libraries were sequenced as 45 bp single-end reads at ˜40 million reads per library using Illumina NextSeq 500 platform (Illumina).
RNA-sequencing (RNA-seq), m6A methylated RNA immunoprecipitation sequencing (m6A MeRIP-seq) and YTHDF1 RNA immunoprecipitation sequencing (RIP-seq) reads were aligned to the human reference genome hg38 by using STAR (version 2.4.2a) ma with the reference annotation GENCODE version 25.m5 HTSeq/DESeq2 pipeline was then applied to quantify and normalize the IP and INPUT per each gene for each type of data.m6,m7 To identify the differential expressed genes (DEGs) from RNA-seq data, the DESeq2 was employed with the criteria that abs (log2 (shYTHDF1/shControl))>0.58 and FDR<0.05. m6A MeRIP-seq uniquely mapped reads for every sample were subsampled to the same read depth (30 million), and reads without duplication were used for peak calling by MeTPeakm3. For the identification of the m6A peak summit and motif discovery, in-house scripts and DREME (MEME Suite: version 4.11.2.1) were used.m8 The peak summit-based m6A distributions along mRNA transcripts were performed by Guitar (version 1.20.1).m2 For RIP-seq data, the genes bound by YTHDF1 were defined as genes with enrichment fold change (log2 (IP/INPUT)) greater than 1.m10 Functional enrichment analysis was performed through the web server g: Profilerm11 using annotated genes as background. The R package clusterProfiler (version 3.10.1) was used for pre-ranked GSEA based on log2 (RNA levels fold change)×−log10 (p value).m12
Active GTP-bound RhoA levels were measured using RhoA Pull-Down Activation Assay Biochem Kit (Cytoskeleton). Briefly, cell lysates were incubated with rhotekin-RBD beads for 1 h at 4° C. The protein/beads complexes were washed with the provided wash buffer and the bound proteins were eluted in 2X Bolt™ LDS Sample Buffer (Thermo Fisher Scientific) and then analyzed by Western blot.
ROCK activity was determined by 96-Well ROCK Activity Assay Kit (Cat. #STA-416, Cell Biolabs) following the manufacturer's instructions. Briefly, 8 μg of total protein was added in each well and incubated at 30° C. for 1 h. After adding Stop Solution, the absorbance at 450 nm was measured.
CRC cells were transfected with ARHGEF2 siRNAs or control siRNA (Thermo Fisher Scientific). 24 h post transfection, the cells were seeded at ˜5000 cells per well in 96-well plates. Two days after seeding, cell viability was assessed by MTT assay (Cat. #M6494; Thermo Fisher Scientific) following the manufacturer's instructions. In addition, 48 h after siRNA transfection, the CRC cells were seeded into the upper chambers of Corning® 6.5 mm Transwell® with 8.0 μm Pore Polycarbonate Membrane Insert, Sterile (Cat. #3422, Corning) for analysis of cell migration.
Lipid Nanoparticle (LNP) Formulation of siRNA Drug
siRNA-loaded LNP formulations were formed using microfluidic rapid mixing method as previously reported.m13 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and 1,2-dimyristoyl-rac-glycero3-methoxy (poly(ethylene glycol))-2000 (DMG-PEG2000), were purchased from Avanti Polar Lipids, Inc (Alabaster, AL). DLin-MC3-DMA was purchased from Organix, Inc. (Woburn, MA). Lipids were mixed in ethanol at a molar ratio of DLin-MC3DMA/DSPC/Cholesterol/DMG-PEG2000: 50/10/38.5/1.5. 2′-O-Methyl (2′-OMe) modified siRNA was dissolved in a 25 mM acetate buffer (pH=4.0). The two phases were mixed through herringbone microfluidic chips (microfluidic ChipShop, Germany) at a volumetric flow rate ratio of 3:1 (aqueous to ethanol). The mixed solution was dialyzed against PBS overnight to remove the ethanol and change external pH to 7.4. Afterwards the formulations were passed through a 0.22 μm filter before use. The hydrodynamic size and dispersity of LNP was characterized by a Zetasizer Nano ZS (Malvern Instruments, United Kingdom). siRNA encapsulation efficiency was measured by Ribogreen Assay (Thermo Fisher Scientific).
All sequencing data of this study have been deposited in NCBI's Gene Expression Omnibus (GEO) database under accession number GSE159425 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159425; reviewer token: qjifuqesdbavtwn).
Data are presented as mean±standard deviation (SD). The independent Student's t test was used to compare the difference between 2 preselected groups. The chi-square test was used for comparison of incidence. The difference in growth rate between the two groups was determined by repeated measures analysis of variance (ANOVA). Value of P<0.05 was taken as statistical significance.
YTHDF1 is Amplified in CRC and Associated with Metastatic Gene Signature
We first analyzed mRNA expression of m6A regulators in the TCGA CRC dataset, revealing that YTHDF1 is the most highly up-regulated one in CRC compared to adjacent non-tumor tissues (
YTHDF1 is located in 20q13.31, a region that is frequently amplified in CRC. To determine if YTHDF1 is up-regulated via gene amplification in CRC, we first analyzed the YTHDF1 copy number variations (CNVs) in our CRC cohort (N=88; stage III and IV) using a specific TaqMan probe against YTHDF1, and found that YTHDF1 copy number gain (3˜4 copies) and gene amplification (>5 copies) were present in 55.4% (49/88) and 13% (11/88) of CRC tumors, respectively (
Amplification and upregulation of YTHDF1 in CRC compared to non-tumor tissues indicate its function in CRC tumorigenesis. We further compared the gene expression profiles of CRCs from the TCGA cohort with or without YTHDF1 gain/amplification using Gene Set Enrichment Analysis (GSEA) (data not shown). Among all the gene sets depleted in CRC tumors with YTHDF1 gain/amplification as compared to those YTHDF1 diploid tumors, the ‘RICKMAN Metastasis DN’ gene signature (genes down-regulated in metastatic versus non-metastatic head and neck squamous cell carcinoma) was the most enriched gene set (
To determine the tumorigenic function of YTHDF1 in CRC, we generated Ythdf1 knockout mice by CRISPR/Cas9. CRISPR/Cas9-mediated deletion resulted in a frameshift and a premature stop codon in the exon 4 of mouse Ythdf1 gene, generating a truncated protein lacking the YTH domain and triggering nonsense-mediated mRNA decay (NMD) (
We next determined the function of YTHDF1 in human CRC cell lines and patient-derived CRC organoids. YTHDF1 knockdown suppressed cell growth of HCT116 and HT-29 in vitro (
We next investigated the role of YTHDF1 in the pro-metastatic capacity of CRC cells. Transwell assay showed that YTHDF1 knockdown evidently decreased CRC cell migration and invasion in vitro, whereas cell migration and invasion abilities of CRC cells were significantly increased upon YTHDF1 overexpression (
To elucidate the molecular mechanism underlying the oncogenic role of YTHDF1, we performed RNA-sequencing (RNA-seq) in YTHDF1-knockdown and control HCT116 cells (
m6A deposition influences RNA processing in the nucleus (e.g. splicing and export), and RNA decay and translation in the cytoplasm.21,22 YTHDF1 is a m6A reader that binds to the m6A-modified mRNA via YTH domain to facilitate translation in cytoplasm in Hela cells12. We thus examined subcellular localization of YTHDF1 by immunofluorescence (IF) staining in CRC cells. YTHDF1 is localized in cytoplasm in both untreated and nuclear export inhibitor Leptomycin B (LMB)-treated HCT116 cells (
m6A MeRIP-seq showed that “GGAC” was the most enriched motif found in m6A peaks identified from both YTHDF1-knockdown and control HCT116 cells (
ITRAQ (isobaric tagging for relative and absolute quantification)-based proteomic profiling of YTHDF1-knockdown and control HCT116 cells were performed to identify differentially expressed proteins.24 Three experimental replications were performed for each group. 3,222 proteins were detected in at least two out of three replicates per group in all shYTHDF1 and control groups, and 214 differentially expressed proteins were identified in both shYTHDF1 groups compared to control (
Amongst the 6 key YTHDF1 targets, ARHGEF2 belongs to the regulators of Rho family of GTPases, one of the top pathways enriched in YTHDF1 targets (
Binding of YTHDF1 to ARHGEF2 overlapped with m6A peaks in the 3′-untranslated region (UTR) near the stop codon of ARHGEF2 mRNA (
ARHGEF2 functions as a RhoA-specific guanine nucleotide exchange factor (GEF) for Rho GTPase, activating RhoA signaling to promote the induction of stress fibers and focal adhesions.27-30 Consistently, YTHDF1-knockdown cells displayed lowered levels of active RhoA (
Rescue assays were performed to confirm whether ARHGEF2 is indispensable for YTHDF1-induced oncogenic transformation. ARHGEF2 re-expression in YTHDF1-knockdown HCT116 cells significantly restored cell proliferation and invasion in vitro (
Since our data demonstrated that ARHGEF2 is a key functional target of YTHDF1, we next asked if targeting ARHGEF2 was more effective in CRC cells with high YTHDF1 expression. ARHGEF2 knockdown led to significantly decreased cell growth and migration abilities in YTHDF1-high CRC cell lines (HCT116, RKO and SW1116) but not in YTHDF1-low CRC cell lines (DLD-1, LoVo and LS 180) (
Targeting ARHGEF2 Using LNP siRNA for CRC Therapy
siRNA nanoparticles with improved biocompatibility have shown attractive potential for disease treatments as theoretically any gene could be targeted by this approach.31 Since no drug is available to directly target ARHGEF2, we used a Food and Drug Administration (FDA) approved LNP formulation technology for siRNA delivery to target ARHGEF2.32 To improve in vivo stability, all pyrimidine bases (C/U) in both strands of siRNAs were modified by 2′-O-Methyl (2′-OMe).33 siRNA encapsulation efficiency (siCtrl: 91.6%; siARHGEF2-1:93.7%) and the hydrodynamic size (siCtrl: 65.38±0.50 nm, Polydispersity Index (PDI): 0.112; siARHGEF2-1:60.45±2.35 nm, PDI: 0.05) and dispersity of LNP (
It has been reported that LNP siRNA is well distributed throughout the subcutaneous xenograft hours after intratumoral injection and the knockdown effect of a single dose could last for up to 4 days.34 Therefore, in our xenograft model, after subcutaneous tumor volume reached approximately 100 mm3, the mice were randomly assigned for treatment with LNP siCtrl or LNP siARHGEF2-1 (2 mg/kg on day7, day9, day11) every two days via intratumoral injection for 7 days. Consistent with in vitro experiments (
Liver is the most common site of metastasis for CRC patients. Over 50% of CRC patients develop liver metastases over the course of their lives, and approximately 14%˜18% of CRC patients have liver metastases at their initial medical consultation.35,36 Reducing liver metastases would be of great importance for CRC patients. Thus, the high accumulation of Onpattro LNP in the liver prompted us to test the potential of LNP siARHGEF2-1 for inhibiting liver metastases in vivo following intravenous administration.32 The mouse liver metastasis model was established by injecting 7.5×105 HCT116 cells intrasplenically into each NOD/SCID mouse. Twelve days after surgery, the mice were randomly assigned for treatment with LNP siCtrl or LNP siARHGEF2-1 every four days (2 mg/kg on day12, day16, day20, day24) via tail-vein injection for 16 days (
Emerging evidence implies crucial roles of m6A epitranscriptome in every hallmark of cancer biology.9 In this study, we identified YTHDF1 as the most highly up-regulated m6A regulator in CRC, implying its role in converting deregulated m6A modifications to pro-tumorigenic signals. Noticeably, YTHDF1 expression in human CRC is correlated with metastatic progression. Using Ythdf1 knockout mice, CRC cell lines and primary CRC organoids, we demonstrated that YTHDF1 exerts pro-tumorigenic effects by enhancing tumor growth, migration/invasion and metastasis.
YTHDF1 is a cytoplasmic m6A reader that affects protein translation via interacting with translation machinery.12 In this regard, multiomics integration analysis was performed to characterize key YTHDF1 target(s) responsible for its pro-tumorigenic functions. We pinpointed ARHGEF2 for further investigation. We validated YTHDF1-m6A-ARHGEF2 axis as a critical molecular signaling involved in YTHDF1-mediated tumorigenesis and metastasis in vitro and in vivo. ARHGEF2 functions to activate RhoA signaling as a RhoA-specific GEF for Rho GTPase, and it mainly participates in cytoskeleton dynamics, focal adhesion and stress fibre formation under physiological conditions.28,37 In support of our data, ARHGEF2 has been reported to promote cell growth and survival by regulating cell cycle transition, apoptosis and transformation.27,38 Pro-tumorigenic effects of ARHGEF2 involved both RhoA-dependent and -independent mechanisms, such as induction of RAS/MAPK.38
Recent studies have uncovered alternative targets of YTHDF1 in human cancers. For example, YTHDF1 has been shown to modulate the canonical WNT/β-catenin signaling through affecting the translation efficiency of upstream WNT regulators FZD9 and WNT6 in human CRC cells, as well as β-catenin transcriptional partner TCF7L2 in mice intestines.39,40 However, those proteins were not detected in our proteomic dataset or the CPTAC CRC cohort, which may be due to low expression of those proteins in CRC. Although it is likely that YTHDF1 regulates multiple signal pathways, the fact that ARHGEF2 overexpression can rescue gene expression and phenotypes of YTHDF1 knockdown suggests it is a main target.
MSI-H/dMMR CRC subtype accounts for only 5% of metastatic CRC.41 YTHDF1 is mainly amplified in non-MSI-H/dMMR CRC patients (data not shown), which comprise 95% of metastatic CRC cases and don't respond to immunotherapy and have limited options for targeted therapy.42 Therefore, targeting the YTHDF1-m6A-ARHGEF2 axis might be a promising therapeutic approach. The recent key advances in LNP delivery technology with efficient encapsulation of siRNA and favorable pharmacokinetics and safety highlighted the potential of developing LNP siRNA drugs targeting YTHDF1-m6A-ARHGEF2 axis.32 The LNP siARHGEF2 system was shown to have an efficient targeting function in vitro and its potency was validated in the xenograft and liver metastasis model. Collectively, the LNP siARHGEF2 drug may provide a potential therapeutic option for metastatic CRC patients through modulation of the epitranscriptome.
In summary, m6A reader YTHDF1 promotes CRC tumorigenesis and metastasis through up-regulation of ARHGEF2 translation and protein expression. Given the widespread YTHDF1 upregulation in CRC and its pro-oncogenic role in CRC pathogenesis, targeting YTHDF1-m6A-ARHGEF2 axis could be a promising therapeutic strategy for inhibition of CRC progression and metastasis. Finally, our work highlights the potential of LNP siARHGEF2 drug for tumor treatment.
Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the following reference list, are incorporated by reference.
This application claims priority to U.S. Provisional Application No. 63/299,054 filed on Jan. 13, 2022, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2023/050034 | 1/12/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63299054 | Jan 2022 | US |
