Patent Application
20230123585
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 6,151 Byte ASCII (Text) file named “2021-03-03_38142-601_SQL_ST25.txt,” created on Mar. 3, 2021.
The present disclosure provides novel materials and methods related to the treatment of cancer. In particular, the present disclosure provides compositions and methods for treating and/or preventing cancer based on the attenuation of Methyltransferase-like Protein 3 (METTL3) activity in a tumor cell. The compositions and methods disclosed herein include the use of a deubiquitinase inhibitor with or without an agent that modulates chromatin state and/or an agent that modulates DNA damage repair.
Diffuse Embryonic stem cells (ESCs) are derived from the inner cell mass of the pre-implantation blastocyst. Under appropriate in vitro culture conditions, ESCs proliferate indefinitely without differentiation, a property referred to as self-renewal, and at the same time retain the developing potential to generate cells of three primary germ layers, known as pluripotency. Studies of ESCs hold promise for tissue repair and provide a potential tool for modeling human disease. In order to fulfill the potential of ESCs, it is critical to understand how ESCs are regulated. The differentiation depends on many regulators that control gene expression, including DNA methylation, transcription factors, and histone/RNA modifications. Mouse, rat, and human ESCs, for instance, share a common subset of transcription factors that specify “stemness”, which include Oct4, Sox2, and Nanog. Signaling pathways have also been shown to regulate ESC fate determination. The JAK/STAT3, ERK, Wnt, and TGF pathways all play roles in affecting downstream gene regulation. For example, ERK signaling guides ESCs to exit pluripotency by phosphorylation of transcription factors that ultimately inhibit expression of genes that maintain pluripotency. Current efforts further seek to elucidate the molecular regulators and signaling pathways that maintain proper differentiation of ESCs.
Recent studies have shown that messenger RNA (mRNA) modifications play a critical role in regulating stem cell differentiation and animal development. Among over 150 known RNA modifications, N6-methyladenosine(m6A) is an evolutionarily conserved and the most abundant internal mRNA modification in most eukaryotic mRNA. m6A is reversibly, site-selectively installed on mRNA transcripts by “writers,” with a portion that can be removed by “erasers.” The m6A methyltransferase “writer” complex has a heterodimeric core made up of the catalytic component METTL3 and its binding partner METTL14; it also includes a co-factor WTAP (Wilms' Tumor 1-Associating Protein). Meanwhile, “eraser” proteins FTO and ALKBHS remove the m6A modification.
m6A can not only affect RNA secondary structure, but also be recognized by m6A “reader” proteins, which exert effects on mRNA metabolism and translation. These m6A-dependent functions include translation initiation, RNA decay, and splicing. It is not surprising, then, that m6A has emerged as a main regulator of gene expression, particularly during development and cell differentiation. In particular, METTL3 has been found to play an essential role in early development. Loss of METTL3 in mouse embryonic cells depletes m6A and increases stability of certain transcripts such as Nanog. This impedes decay of pluripotency factors that maintain self-renewal, thereby also delaying proper lineage priming and fate transition, leading to early embryo lethality. Depletion of the Drosophila METTL3 homolog Ime4 prevents proper Sexually splicing and thus leading to failure of sex determination. These studies have shown that m6A methylation controls stability of transcripts, including those that promote naïve pluripotency and require timely downregulation for proper differentiation, and that m6A deposition is crucial for the temporal regulation of development. The importance of m6A methylation has been well described recently, yet gaps in the understanding of how this process is regulated remain.
Embodiments of the present disclosure include a composition for attenuating Methyltransferase-like Protein 3 (METTL3) in a tumor cell. In accordance with these embodiments, the composition includes at least one deubiquitinase inhibitor, at least one chromatin state modulator and/or at least one DNA damage modulator, and a pharmaceutically acceptable carrier or excipient.
In some embodiments, the at least one deubiquitinase inhibitor targets Ubiquitin Carboxyl-terminal Hydrolase 5 (USP5). In some embodiments, the at least one chromatin state modulator includes a bromodomain and extraterminal domain (BET) inhibitor, a histone methyl transferase (HMT) inhibitor, and/or a poly(ADP-ribose) polymerase 1 (PARP1) inhibitor. In some embodiments, the at least one DNA damage repair modulator induces DNA damage and/or inhibits DNA repair.
In some embodiments, inhibiting USP5 attenuates METTL3 protein stability and/or activity.
In some embodiments, the at least one deubiquitinase inhibitor comprises EOAI3402143, vialinin, WP1130, mebendazole, PYR-41, gossypetin, formonectin, suramin, and combinations thereof. In some embodiments, the BET inhibitor comprises a thienotriazolodiazepine, OTX015, BET-d246, ABBV-075, I-BET 151, I-BET 762, CPI 203, PFI-1, RVX-208, Dinaciclib, and combinations thereof. In some embodiments, the thienotriazolodiazepine is JQ1.
In some embodiments, the HMT inhibitor comprises chaetocin, GSK343, UNC199, SGC0946, F5446, Pinometostat, EPZ004777, EPZ005687, tazemestostat, JQEZ5, CPI-1205, EPZ001989, EBI-2511, PF-06726304, El 1, GSK503, GSK126, CPI-169, ZLD 1039, SAH-EZH2, NSC 617989, CPI-169, CPI-360, EPZ6438, and combinations thereof.
In some embodiments, the PARP inhibitor comprises olaparib, rucaparib, veliparib, talazoprib, AG-14361, INO-1001, A-966492, PJ34, Niraparib, UPF 1069, ME0328, Pamiparib, NMS-P118, E7449, picolinamide, Benzamide, Nu1025, Iniparib, AZD2461, BGP-15, and combinations thereof.
In some embodiments, the at least one DNA damage repair modulator comprises bleomycin, 5-FU, ceralasertib (AZD6738), cisplatin, oxaliplatin, carboplatin, Cytoxan, and combinations thereof.
In some embodiments, the composition comprises at least one deubiquitinase inhibitor and wherein the at least one chromatin state modulator is a BET inhibitor. In some embodiments, the composition comprises at least one deubiquitinase inhibitor and wherein the at least one chromatin state modulator is a HMT inhibitor. In some embodiments, the composition comprises at least one deubiquitinase inhibitor and wherein the at least one chromatin state modulator is a PARP inhibitor. In some embodiments, the composition comprises at least one deubiquitinase inhibitor and at least one DNA damage repair modulator.
Embodiments of the present disclosure also include a method of treating or preventing cancer in a subject comprising administering any of the pharmaceutical compositions described above. In some embodiments, a method of treating or preventing cancer in a subject includes administering a composition comprising at least one deubiquitinase inhibitor, and at least one of a bromodomain and extraterminal domain (BET) inhibitor, a histone methyl transferase (HMT) inhibitor, a poly(ADP-ribose) polymerase 1 (PARP1) inhibitor, and/or a DNA damage repair modulator.
In some embodiments of the method, the composition further comprises a pharmaceutically acceptable carrier or excipient, and wherein the composition is administered to a subject diagnosed with cancer.
In some embodiments of the method, the at least one deubiquitinase inhibitor comprises EOAI3402143, vialinin, WP1130, mebendazole, PYR-41, gossypetin, formonectin, suramin, and combinations thereof.
In some embodiments of the method, the BET inhibitor comprises a thienotriazolodiazepine, OTX015, BET-d246, ABBV-075, I-BET 151, I-BET 762, CPI 203, PFI-1, RVX-208, Dinaciclib, and combinations thereof.
In some embodiments of the method, the HMT inhibitor comprises chaetocin, GSK343, UNC199, SGC0946, F5446, Pinornetostat, EPZ004777, EPZ005687, tazemestostat, JQEZ5, CPI-1205, EPZ001989, EBI-2511, PE-06726304, El1, GSK503, GSK126, CPI-169, ZLD 1039, SAH-EZH2, NSC 617989, CPI-169, CPI-360, EPZ6438, and combinations thereof.
In some embodiments of the method, the PARP inhibitor comprises olaparib, rucaparib, veliparib, talazoprib, AG-14361, INO-1001, A-966492, PJ34, Niraparib, UPF 1069, ME0328, Pamiparib, NMS-P118, E7449, picolinamide, Benzamide, Nu1025, Iniparib, AZD2461, BGP-15, and combinations thereof.
In some embodiments of the method, the composition attenuates METTL3 stability and/or activity and induces apoptosis of a cancer cell.
In some embodiments of the method, the composition comprises at least one deubiquitinase inhibitor and at least one BET inhibitor. In some embodiments of the method, the combination of the at least one deubiquitinase inhibitor and the at least one BET inhibitor exhibits a synergistic effect on cancer cell viability.
In some embodiments of the method, the composition comprises at least one deubiquitinase inhibitor and at least one HMT inhibitor. In some embodiments of the method, the combination of the at least one deubiquitinase inhibitor and the at least one HMT inhibitor exhibits a synergistic effect on cancer cell viability.
In some embodiments of the method, the composition comprises at least one deubiquitinase inhibitor and at least one PARP inhibitor. In some embodiments of the method, the combination of the at least one deubiquitinase inhibitor and the at least one PARP inhibitor exhibits a synergistic effect on cancer cell viability.
In some embodiments of the method, the combination of the at least one deubiquitinase inhibitor and the at least one DNA damage repair modulator or inhibitor that blocks DNA damage repair exhibits a synergistic effect on cancer cell viability.
In some embodiments of the method, the cancer is selected from the group consisting of melanoma, breast cancer, lung cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, colorectal cancer, renal cancer, skin cancer, head & neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, glioblastoma, lymphoma, and leukemia.
The present disclosure relates to the treatment and/or prevention of cancer. In particular, the present disclosure provides novel compositions and methods for treating and/or preventing cancer based on the attenuation of Methyltransferase-like Protein 3 (METTL3) activity in a tumor cell. In accordance with these embodiments, the present disclosure provides compositions and methods involving the use of a deubiquitinase inhibitor with or without an agent that modulates chromatin state and/or an agent that modulates DNA damage repair.
Generally, m6A RNA methylation plays substantial roles in regulating RNA metabolism and, in doing, so, tunes gene expression and controls biological functions. The modification is installed by the METTL3/METTL14 heterodimeric complex, and can be reversed by the two demethylases. While many studies have shown the importance of METTL3 in cancer, stem cell, and other physiology, few have shown how METTL3 itself is post-translationally regulated.
Embodiments of the present disclosure identify an ERK2-METTL3/WTAP signaling axis that regulates mESC differentiation and potentially affect tumorigenesis. Initially, a genome-wide CRISPR screen was deployed using an m6A methylation-dependent GFP reporter. Ras and MAPK pathway were identified as the top pathways in the positive regulation of m6A methylation. Biochemical studies showed that ERK proteins could phosphorylate METTL3 on S43/S50/S525 and WTAP at S306/S341. It was also found that phosphorylation of METTL3 decreases METTL3 ubiquitination through interaction with USP5. These findings explain elevated m6A levels on polyA-tailed RNA upon ERK activation. This pathway underlines a previously unrecognized effect of ERK activation through RNA methylation during differentiation in pluripotent mouse ESCs (see, e.g.,
Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
“Coefficient of variation” (CV), also known as “relative variability,” is equal to the standard deviation of a distribution divided by its mean.
“Controls” as used herein generally refers to a reagent whose purpose is to evaluate the performance of a measurement system in order to assure that it continues to produce results within permissible boundaries (e.g., boundaries ranging from measures appropriate for a research use assay on one end to analytic boundaries established by quality specifications for a commercial assay on the other end). To accomplish this, a control should be indicative of patient results and optionally should somehow assess the impact of error on the measurement (e.g., error due to reagent stability, calibrator variability, instrument variability, and the like).
“Correlated to” as used herein refers to compared to.
“Sample,” “test sample,” “specimen,” “sample from a subject,” and “patient sample” as used herein may be used interchangeably and may be a sample of blood, such as whole blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.
“Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal and a human. In some embodiments, the subject may be a human or a non-human. The subject or patient may be undergoing other forms of treatment.
“Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, llamas, camels, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, rabbits, guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
“Treat,” “treating” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease and/or injury, or one or more symptoms of such disease, to which such term applies. Depending on the condition of the subject, the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Such prevention or reduction of the severity of a disease prior to affliction refers to administration of a pharmaceutical composition to a subject that is not at the time of administration afflicted with the disease. “Preventing” also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease. “Treatment” and “therapeutically,” refer to the act of treating, as “treating” is defined above.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
Embodiments of the present disclosure pertain to the finding that ERK-mediated phosphorylation of METTL3 is important for downregulation of m6A-labeled pluripotency transcripts in order to induce mESC differentiation. Consistent with previous observations, m6A-seq data revealed extensive mRNA m6A methylation in mESCs. Additionally, as described further herein, upon loss of METTL3/WTAP phosphorylation, differentially methylated transcripts are enriched for genes involved in pluripotency, RNA processing, and development (similar to those found in METTL3 KO/KD studies). This supports a model in which METTL3 phosphorylation is necessary for regulation of pluripotency and differentiation.
Results provided here further explain the importance of METTL3 in regulating gene expression that leads to mESC state transitions. For example, Mettl3-deficient mESCs fail to exit pluripotency despite differentiation cues, at least in part because m6A destabilizes transcripts that promote pluripotency. Previous studies have shown distinct effects of Mettl3 removal between the hyper-naive state and primed naive state, the former towards promoting pluripotency and the latter differentiation. Results provided herein suggest that ERK activation may further increase m6A methylation on key pluripotent transcripts, thus contributing to their decay. Tuning the phosphorylation state of METTL3 may be an effective post-translational mechanism to adjust global mRNA m6A methylation upon signaling or stress response.
While the results provided herein indicate that phosphorylation of METTL3 affects interaction with WTAP and USP5, the effects of other signaling pathways or binding partners affected. For example, the TGF-β signaling pathway component SMAD2/3 interacts with the METTL3/METT14/WTAP complex to promote m6A binding to particular transcripts in mESCs. ZFP217 has also been found to interact with and sequester METTL3, thereby restricting m6A methylation of certain transcripts in ESCs. Knockdown of ZFP217 results in decreased lifetime of pluripotency transcripts as well. WTAP was also found to be phosphorylated by ERK. In HEK293T cells, expression of MEK, HER, or B-RAF could increase association between METTL3 and WTAP. In smooth muscle cells, IGF-1, which transmits signals along the MAPK and PI3K pathways, induces degradation of WTAP protein. This effect is mediated by the PI3K/AKT pathway, and modulation of the ERK pathway had no effect.
Genome integrity is constantly under challenge by cellular and environmental factors. DNA damage response (DDR) can detect and repair damaged DNA, and suspend cell division until the repair is complete. Studies over the last decades have emphasized the roles of chromatin components in response to DNA damage. For example, at an early stage of DDR, histone marks are installed at specific regions to make them more accessible to repair factors and to inhibit transcription from a damaged template. Despite these advances, key factors involved in DNA damage repair remain to be uncovered. Previous work has shown that m6A methylation is transiently induced at DNA damage sites in response to UV irradiation, and that m6A facilitates Pol lc recruitment to damage sites to ensure efficient DNA repair and cell survival. However, the underlying mechanism is not clear.
However, m6A methylation of chromosome-associated regulatory RNAs (carRNAs), in particular promoter-associated RNA (paRNA), enhancer RNA (eRNA), and repeats RNAs by METTL3 controls their stability on the chromatin. And depletion of METTL3 in mouse embryonic stem cells (mESCs) elevates levels of carRNAs and promotes open chromatin state and downstream gene transcription in mESC2. Therefore, carRNA m6A methylation may impact DNA damage repair.
For example, a rapid increase of chromatin openness in mESCs was previously observed upon UV irradiation, followed by a reversal back to the normal level; however, in Mettl3 knockout mESCs, the increased chromatin accessibility induced by UV damage could not reverse back after 2 hours (see, e.g.,
The results described in the present disclosure demonstrate that METTL3 modulates dsDNA damage repair signaling (e.g., homologous recombination (HR) and non-homologous end joining (NHEJ) pathways); therefore, inhibition of METTL3 will affect tumors associated with chromosome and microsatellite instability and/or DNA damage repair defect (e.g., BRCA1/BRCA2 mutations, DNA mismatch repair mutations, p53 mutations, and the like). METTL3 modulation is thus a target for anti-cancer therapies, including but not limited to, therapies designed to target METTL3 directly, indirectly (e.g., USP5 modulation), and/or in combination with other agents that modulate chromatin state or DNA damage repair.
In accordance with these embodiments, the compositions and methods provided herein can target major repair pathways and key proteins used to process the various types of DNA damage. In non-homologous end-joining (NHEJ), for example, the Ku70/Ku80 complex binds to the DNA double-strand break ends and recruits the other indicated components. In base-excision repair (BER), poly(ADP-ribose) polymerase-1 (PARP-1) detects and binds to single-strand breaks and ensures accumulation of other repair factors at the breaks. Single-strand breaks containing modified DNA ends are recognized by damage-specific proteins such as apurinic/apyrimidinic endonuclease (APE1), which subsequently recruits Polβ and XRCC1-DNA ligase Ma to accomplish the repair. The proteins involved in these pathways have been shown to be dysregulated in various types of cancers, and METTL3 inhibition can be used to target one or more of them to treat and/or prevent cancer; these targets include, but are not limited to, PARP-1, APE1, XRCC1, DNA ligase III, Ku70/Ku80, DNA-PK, Artemis, XRCC4, DNA ligase IV, XLF, RPA, BRCA1, BRCA2, PALB2, and RAD51, among others.
For example, and with regard to NHEJ specifically, inhibitors of DNA-PK, including NU7026 and NU7441, were found to induce extreme sensitivity to ionizing radiation as well as DNA-damaging agents in preclinical studies. The dual mTOR and DNA-PKcs inhibitor CC-115 is undergoing early clinical evaluation. KU-0060648 is a potent dual inhibitor of DNA-PK and PI-3K, which has recently been reported to enhance etoposide and doxorubicin.
In another example, inactivation of DNA damage response proteins is also observed in various cancers. The p53 gene is one of the most frequently mutated genes in human sporadic cancers. Although the reported frequencies of p53 mutations vary among the types of cancer, it is estimated that more than half of cancers might have inactivated p53 due to mutations, deletions, loss of heterozygosity of the gene, or decreased expression. Although inactivating mutations in ATM, BRCA1, or BRCA2 are less frequent than those in the p53 gene, decreased expression of ATM, the MRN complex, Chk2, RAD51, BRCA1, BRCA2, and ERCC1 is frequently observed. Promoter hypermethylation of the BRCA1 gene has also been observed and may be one of the predominant mechanisms for deregulation of the BRCA1 gene.
In another example, ATM and the MRN complex, which act as sensors or mediators in the DNA damage response, have been considered to be targets for cancer therapy, and several promising ATM inhibitors have been developed. KU55933, for example, is the first specific inhibitor of ATM, and it inhibits radiation-induced ATM-dependent phosphorylation events and sensitizes cancer cells to radiation and topoisomerase inhibitors. KU60019, an improved analog of KU55933, inhibits the DNA damage response and effectively radiosensitizes human glioma cells. Mirin is an inhibitor of the MRN complex, which prevents MRN-dependent activation of ATM without affecting ATM protein kinase activity and inhibits MRE11-associated exonuclease activity. Telomelysin is another inhibitor that inhibits the MRN complex through the adenoviral E1B-55 kDa protein. Additionally, schisandrin B was recently identified as a moderate selective ATR inhibitor (may also affect ATM at high concentrations). Recently, two novel ATR inhibitors, NU6027 and VE-821, were also shown to sensitize cells to a variety of DNA-damaging agents in preclinical studies.
Taken together, and as described further herein, mechanisms related to METTL3 inhibition can be used alone or in combination with the various modulators targeting DNA repair pathways and key proteins used to process the various types of DNA damage. Thus, embodiments of the present disclosure include a composition for attenuating METTL3 in a tumor cell. In accordance with these embodiments, the composition includes at least one deubiquitinase inhibitor, at least one chromatin state modulator and/or at least one DNA damage modulator, and a pharmaceutically acceptable carrier or excipient.
In some embodiments, the at least one deubiquitinase inhibitor targets Ubiquitin Carboxyl-terminal Hydrolase 5 (USP5). In some embodiments, the at least one chromatin state modulator includes a bromodomain and extraterminal domain (BET) inhibitor, a histone methyl transferase (HMT) inhibitor, and/or a poly(ADP-ribose) polymerase 1 (PARP1) inhibitor. In some embodiments, the at least one DNA damage repair modulator induces DNA damage and/or inhibits DNA repair. In some embodiments, inhibiting USP5 attenuates 1METTL3 protein stability and/or activity.
In some embodiments, the at least one deubiquitinase inhibitor comprises EOAI3402143, vialinin, WP1130, mebendazole, PYR-41, gossypetin, formonectin, suramin, and/or any combinations thereof. In some embodiments, the BET inhibitor comprises a thienotriazolodiazepine, OTX015, BET-d246, ABBV-075, I-BET 151, I-BET 762, CPI 203, PFI-1, RVX-208, Dinaciclib, and/or any combinations thereof. In some embodiments, the thienotriazolodiazepine is JQ1.
In some embodiments, the HMT inhibitor comprises chaetocin, GSK343, UNC199, SGC0946, F5446, Pinometostat, EPZ004777, EPZ005687, tazemestostat, JQEZ5, CPI-1205, EPZ001989, EBI-2511, PF-06726304, El1, GSK503, GSK126, CPI-169, ZLD 1039, SAH-EZH2, NSC 617989, CPI-169, CPI-360, EPZ6438, and/or any combinations thereof.
In some embodiments, the PARP inhibitor comprises olaparib, rucaparib, veliparib, talazoprib, AG-14361, INO-1001, A-966492, PJ34, Niraparib, UPF 1069, ME0328, Pamiparib, NMS-P118, E7449, picolinamide, Benzamide, Nu1025, Iniparib, AZD2461, BGP-15, and/or any combinations thereof.
In some embodiments, the at least one DNA damage repair modulator comprises bleomycin, 5-FU, ceralasertib (AZD6738), cisplatin, oxaliplatin, carboplatin, Cytoxan, and/or any combinations thereof.
In some embodiments, the compositions of the present disclosure comprise at least one deubiquitinase inhibitor and a BET inhibitor. In some embodiments, the composition comprises at least one deubiquitinase inhibitor and an HMT inhibitor. In some embodiments, the composition comprises at least one deubiquitinase inhibitor and a PARP inhibitor. In some embodiments, the composition comprises at least one deubiquitinase inhibitor and at least one DNA damage repair modulator.
Embodiments of the present disclosure also include a method of treating or preventing cancer in a subject. In accordance with these embodiments, the method includes administering any of the pharmaceutical compositions described herein to the subject. In some embodiments, a method of treating or preventing cancer in a subject includes administering a composition comprising at least one deubiquitinase inhibitor, and at least one of a bromodomain and extraterminal domain (BET) inhibitor, a histone methyl transferase (HMT) inhibitor, a poly(ADP-ribose) polymerase 1 (PARP1) inhibitor, and/or a DNA damage repair modulator. In some embodiments of the method, the composition further comprises a pharmaceutically acceptable carrier or excipient.
In some embodiments, the composition is administered to a subject diagnosed with cancer in order to treat the cancer. In some embodiments of the method, the at least one deubiquitinase inhibitor comprises EOAI3402143, vialinin, WP1130, mebendazole, PYR-41, gossypetin, formonectin, suramin, and/or any combinations thereof. In some embodiments of the method, the BET inhibitor comprises a thienotriazolodiazepine, OTX015, BET-d246, ABBV-075, I-BET 151, I-BET 762, CPI 203, PFI-1, RVX-208, Dinaciclib, and/or any combinations thereof. In some embodiments of the method, the HMT inhibitor comprises chaetocin, GSK343, UNC199, SGC0946, F5446, Pinometostat, EPZ004777, EPZ005687, tazemestostat, JQE,Z5, CPI-1205, EPZ001989, EBI-2511, PF-06726304, El1, GSK503, GSK126, CPI-169, ZLD 1039, SAH-EZI-12, NSC 617989, CPI-169, CPI-360, EPZ6438, and/or any combinations thereof. In some embodiments of the method, the PARP inhibitor comprises olaparib, rucaparib, veliparib, talazoprib, AG-14361, INO-1001, A-966492, PJ34, Niraparib, UPF 1069, ME0328, Pamiparib, NMS-P118, E7449, picolinamide, Benzamide, Nu1025, Iniparib, AZD2461, BGP-15, and/or any combinations thereof.
In some embodiments of the method, the composition attenuates METTL3 stability and/or activity and induces apoptosis of a cancer cell. In some embodiments of the method, the composition comprises at least one deubiquitinase inhibitor and at least one BET inhibitor. In some embodiments of the method, the combination of the at least one deubiquitinase inhibitor and the at least one BET inhibitor exhibits a synergistic effect on cancer cell viability.
In some embodiments of the method, the composition comprises at least one deubiquitinase inhibitor and at least one HMT inhibitor. In some embodiments of the method, the combination of the at least one deubiquitinase inhibitor and the at least one HMT inhibitor exhibits a synergistic effect on cancer cell viability. In some embodiments of the method, the composition comprises at least one deubiquitinase inhibitor and at least one PARP inhibitor. In some embodiments of the method, the combination of the at least one deubiquitinase inhibitor and the at least one PARP inhibitor exhibits a synergistic effect on cancer cell viability. In some embodiments of the method, the combination of the at least one deubiquitinase inhibitor and the at least one DNA damage repair modulator or inhibitor that blocks DNA damage repair exhibits a synergistic effect on cancer cell viability.
In some embodiments of the method, the cancer is selected from the group consisting of melanoma, breast cancer, lung cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, colorectal cancer, renal cancer, skin cancer, head & neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, glioblastoma, lymphoma, and leukemia.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.
The present disclosure has multiple aspects, illustrated by the following non-limiting examples.
ERK Activation Promotes mRNA m6A Methylation. To identify new regulators of m6A RNA methylation, a circular RNA GFP reporter was employed containing a GGACU motif in HeLa cells. The GFP pre-mRNA transcript was assembled by back-splicing to generate a circular RNA that joins two exon fragments of GFP, as depicted in
To determine how the RAS/MAPK pathway could alter m6A methylation, the status of the m6A methyltransferase complex during MAPK pathway activation was investigated. A phos-tag gel revealed that constitutively active MEK S218D/S222D, B-RAF V600E, or HER2 V659E, increased the phosphorylation-dependent mobility shift of METTL3 and WTAP, but not METTL14 (
ERK Phosphorylates METTL3 and WTAP. To determine how ERK activates m6A methylation, it was first tested whether ERK interacts with and phosphorylates the mRNA m6A methyltransferase complex. Co-immunoprecipitation showed that METTL3 associates with ERK1 and ERK2 upon B-RAF transfection (
ERK displays a specificity for phosphorylation at the serine/threonine-proline (S/T-P) motif. Since the S/T-P motif is found in many proteins, ERK either uses a common docking domain (CD) to bind to a D domain (K/R0-2-X1-6-φ-X-φ) or uses the F-site recruitment site (FRS) to bind to the F-site (FX-F/Y-P). Analysis using the Eukaryotic Linear Motif database (http://elm.eu.org) revealed residues 415-421 in METTL3 and residues 71-77 in WTAP as potentially conserved D domains (
Given the physical interaction between ERK and METTL3, and the phosphorylation- based mobility shift induced by the ERK activation, experiments were conducted to identify phosphorylation sites on METTL3. Mass spectrometry analysis showed that ERK phosphorylates METTL3 at three highly conserved residues S43, S50, and S525 (
To investigate METTL3 phosphorylation by ERK inside cells, a polyclonal antibody was generated that targets S43-phosphorylated METTL3. This antibody recognizes S43-phosphorylated METTL3 but not a mutant form of METTL3, METTL3 3A, with all three phosphorylation serine sites replaced with alanine (
To determine the phosphorylation sites of WTAP, experiments were conducted to determine whether mutations of the S/T-P motif affect the ERK-induced phosphorylation. Among the three S/T-P motifs in human WTAP (
USP5 is Required for ERK-Mediated METTL3 Stabilization. Next, experiments were conducted to investigate how ERK-induced phosphorylation increases RNA m6A methyltransferase complex activity. It was observed that ERK activation increased the wild-type (WT) but not 3A METTL3 expression (
Since METTL14 is known to stabilize METTL3, experiments were conducted to investigate whether phosphorylation of METTL3 by ERK affects the METTL3-METTL14 complex formation. The interaction between METTL3 and METTL14 was not obviously affected by ERK inhibition (
To gain further insight into how ERK phosphorylation decreases METTL3 ubiquitination, experiments were conducted to determine if any ubiquitin ligases or deubiquitinases were identified in the CRISPR-based genomic screen. Notably, USP5 was identified as a potential positive regulator (
Phosphorylation of METTL3/WTAP by ERK Facilitates Resolution of Pluripotency. Because both ERK activation and METTL3 expression have been reported to be required for mESCs to exit the pluripotent state upon differentiation, experiments were conducted to investigate whether phosphorylation of METTL3/WTAP affects mESC fate. Wild-type METTL3 and WTAP (R-WT), or non-phosphorylatable METTL3 3A and WTAP 2A (R-3A2A) were re-expressed in homozygous Mettl3 knockout (KO) mESCs (
Mettl3-deficient mESCs fail to exit pluripotency despite differentiation cues, likely because loss of m6A impedes the degradation of pluripotency-promoting transcripts. Experiments were then conducted to examine reported m6A-methylated pluripotency factor transcripts, including Nanog, Zfp42, Klf2, Sox2, and Lefty1. Pou5f 1, which does not harbor m6A modification, was also used as a negative control. m6A-RIP-qPCR confirmed decreased m6A (
Transcripts Affected by Phosphorylation of Methyltransferase Complex in mESCs. To gain further insight into how the phosphorylation of the m6A methyltransferase complex affects the m6A-modified transcripts, the m6A methylome was mapped in mESCs. Comparison of the R-WT with R-3A2A mESCs revealed a global loss of methylation sites (
To expand the observation of pathways or sets of genes that are enriched when comparing R-WT and R-3A2A mESCs, a functional class scoring approach (gene-set enrichment analysis, GSEA) was also performed besides GO analysis. GSEA showed enrichment of histone binding proteins (
Phosphorylation of the m6A Methyltransferase Complex May Affect Tumorigenesis. As one of the most frequently mutated signaling pathways in cancer, the Ras/Raf/MEK/ERK signaling cascade has long been viewed as promising targets for cancer therapy. Given that phosphorylation of the m6A methyltransferase complex by ERK facilitates resolution of pluripotency in mESCs, experiments were conducted to further investigate whether the m6A methyltransferase complex can be similarly regulated in certain cancer cells. METTL3 knockdown is known to induce apoptosis and METTL3 overexpression could promote tumorigenesis in multiple cancer types. Using Cancermine (Lever et al., 2019), a literature-mined resource, it was determined that METTL3 could behave as an oncogene in many cancer types (
Experiments were conducted to first examine melanoma due to the high prevalence of constitutively active BRAF V600E mutation (50-60%) and clinical success with BRAF and MEK inhibitors. The m6A levels on polyA-tailed RNA are higher in the MEL-624 and A375 cells, which harbor a BRAF V600E mutation (
Because knockdown of USP5 increases METTL3 in A375 melanoma cells (
Lastly, considering that HER2 expression phosphorylates METTL3 and WTAP (
Bromodomain and extraterminal domain (BET) family of proteins. The bromodomain and extraterminal domain (BET) family of proteins have been investigated as a potentially effective therapeutic target for treating PDAC tumors. JQ1 inhibits BET protein function by binding to the domain of BET that interacts directly with acetylated lysine residues on specific histones, thereby condensing the chromatin globally and decreasing expression of proteins that rely on BET-dependent mechanisms for transcription. A panel of 9 PDAC cell lines was used to test the anticancer activity of JQ1, and it was found that the inhibition effect of JQ1 varies among different cell types (
The 8902 cell line was used as a JQ1-insensitive cell and the Mia cell as a JQ1-sensitive cell to further investigate the potential synergistic effects of JQ1 and knockdown of METTL3. The results showed that in the JQ1-insensitive cell, JQ1 treatment has a moderate effect on METTL3 RNA level and less of an effect on METTL3 protein level. However, JQ1 treatment after METTL3 knockdown further reduced the polyA RNA m6A, which indicates JQ1 treatment may regulate the accessibility of METTL3 towards its substrate (
Histone methyl transferase (HMT) activity. N6-methyladenosine (m6A), catalyzed by the methyltransferase complex consisting of Mettl3 and Mettl14, is the most abundant RNA modification in mRNAs and participates in diverse biological processes. The mechanisms by which m6A modification affects gene expression are being investigated. In a previous study, gene-set enrichment analysis (GSEA) showed that histone-binding proteins were enriched when comparing m6A-labled genes in wild type and mutant METTL3 mESC. Therefore, an ELISA kit was used to compare 21 different Histone H3 modifications in melanoma (A375) and colon cancer cells (HCT116). As shown in
H3K9me3 and H3K27me3 are repressive chromatin markers that correlate with transcriptional repression. Therefore, a DNaseI-TUENL assay was used to measure chromatin accessibility. As show in
USP5 Inhibition. EOAI is a USP5 inhibitor that could lead to a decreased level of METTL3 protein. When applied together in JQ1-insensitive cells (e.g., 8902 cells), the combination of 5 μmol JQ1 and 1.5 μmol EOAI exhibited comparable effect on cell viability as a single dose of 10 μmol JQ1 or 2.5 μmol EOAI (
USP5 inhibition Leads to Increased Ubiquitination of METTL3. Through performing a CRISPR-based genomic screen using GGACU motif-circular RNA-GFP reporter, USP5 was identified as a potential positive regulator of m6A pathway. It has been shown that mutant B-RAF activates certain deubiquitinases, including USP5. Co-immunoprecipitation showed that the association between METTL3 and USP5 was increased upon B-RAF transfection (
Furthermore, overexpression of METTL3 attenuated, while knockdown of METTL3 sensitized, melanoma and colon cancer cells to USP5 inhibitors EOAI, supporting the connection between USP5 and METTL3 (
Since synergism between METTL3 KD and Ezh2 inhibitors was observed, further experiments were conducted to examine the effect of the combination of the USP5 inhibitor, EOAI, with Ezh2 inhibitors, GSK343 and UCN1999. The results shown in
Similar experiment were conducted to investigate the involvement of poly(ADP-ribose) polymerase 1 (PARP1) in chromatin stability. Considering METTL3 KD affects chromatin status, with possible effects on transcription dynamics, the kethoxal-assisted single-stranded DNA sequencing (KAS seq) was further used to investigate global transcription dynamics. Interestingly, the analysis identified peak loss when METTL3 was knocked down. GO analysis showed the enrichment of DNA damage pathway from the KAS seq. It has been reported that PARP is required to m6A accumulation during DNA damage. Similar experiment were therefore conducted to investigate the involvement of poly(ADP-ribose) polymerase 1 (PARP1) in chromatin stability. The results in
Genomic instability is a characteristic of many human cancers, and could involve defected DNA damage repair. If METTL3-mediated caRNA methylation plays important roles in DNA damage repair, then inhibition of METTL3 could preferentially affect tumors associated with genomic instability. Data provided herein demonstrated that METTL3 knockdown in A375 melanoma cancer cells triggered dsDNA breaks and caused apoptosis (
Next, the potential for synergistic effects with other agents was evaluated. A375 cancer cells were treated with DNA damage agents or inhibitors of specific DNA repair pathways (e.g., DNA damage repair modulators). METTL3 knockdown in A375 cells showed synergistic effects when treated with two DNA damage agents: Bleomycin, an ionizing radiation drug which induces dsDNA breaks (
mESC culture and differentiation. mESCs were generated, maintained, and differentiated essentially as previously described (Geula et al., 2015). METTL3 knockout mESCs were kindly provided by Dr. Howard Y. Chang (Stanford University) and regularly tested negatively for mycoplasma contamination. Established ESC clones were genotyped by PCR and validated as Mettl3-deficient by qPCR and Western blot. mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts in ES medium containing DMEM supplemented with 15% FBS, 1 mM L-glutamine, 0.1 mM mercaptoethanol, 1% non-essential amino acid , 1% Pen/Strep, nucleosides 1,000 U/ml leukemia inhibitory factor, 3 μM CHIR99021 and 1 μM PD0325901. For embryoid body (EB) differentiation, 5×106 ESC were disaggregated with trypsin and transferred to non-adherent suspension culture dishes and cultured in MEF medium (DMEM supplemented with 1% L-Glutamine, 1% Non-essential amino acids, 1% penicillin/streptomycin and 15% FBS) for 8 days.
Cell Culture. HeLa, 293T, 293TN, A375, CHL-1, MEL-624 cells were maintained in DMEM supplemented with 10% FBS and 1% Pen/Strep. MCF-7, T47D, SKBR3, and HCT-116 cells were maintained in RPMI supplemented with 10% FBS and 1% Pen/Strep. BT474 cells were maintained in RPMI supplemented with 20% FBS and 1% Pen/Strep.
Plasmids. The circRNA reporters containing split GFP with a m6A motif were kindly provided by Z. Wang (Chinese Academy of Science, Shanghai, China) and subcloned into pCDH- CMV-MCS-EFlα-RFP (System bioscience, CD512B-1). The CRISPR knockout pooled library (#1000000048), METTL3 (#53739), METTL14 (#53740), WTAP (#53741), pKMyc (#19400), Flag-ATM (#31985), ATR (#31611), Flag-IKKe (#26201), HA-GSK-3β (#14754), ERK1 (#23509), ERK2 (#23498), B-Raf V600E (#17544), pMD2.G (#12259) and psPAX2 (#12260) were ordered from Addgene. Flag-IKKα, Flag-IKKβ, HA-AKT, Flag-mTOR, HA-MEKDD, HA-CDC2, FAK, EGFR, HER2 V659E, HA-ubiquitin, pCMV5-HA, and pCMV5-Flag were kindly provided by M. C. Hung (China Medical University, Taichung, Taiwan). pLightSwtich R01_3′UTR and Nanog 3′UTR were ordered from Switchgear Genomics. Mouse METTL3 (MR209093), mouse WTAP (MR216877), and USP5 (RC224191) were purchased from Origene. METTL3 (human and mice), METTL14, and WTAP were subcloned into pkmyc, METTL14 was subcloned into pCMV5-HA, and WTAP (human and mice), ERK1, ERK2, and USP5 were cloned into pCMV5-Flag. All mutants were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The annealed siMETTL3 (TRCN0000289742) and siUSP5 (TRCN0000306799) specific targeted sequence was inserted into Tet-pLKO (Addgene, #21915). Myc-METTL3-T2A-Flag-WTAP was cloned into pCDH-CuO-MCS-EF 1α-RFP (System Biosciences, QM512B-1). pCDG-EF1α-CymR-T2A-Neo (QM400PA-2) for the cumate suppressor was ordered from System Biosciences.
Transfection and Virus Production. For transient transfection, cells were transfected by Lipofectamine 2000 as previously described (Lee et al., 2007). For lentivirus production, a lentiviral construct (pCDH-CMV-MCS-EF1α-RFP plasmids for overexpressing circRNA-GFP, Tet-pLKO for inducible knockdown of METTL3 or USP5, pCDG-EF1α-CymR-T2A-Neo for cumate repressor, or pCDH-CuO-MCS-EF1α-RFP for inducible overexpression of METTL3-T2A-WTAP) together with pMD2.G and psPAX2 were co-transfected into 293TN cells (System Biosciences). Viruses were concentrated by the PEG-it Virus Precipitation Solution and used for infecting cells in the presence of TransDux (System Bioscience). Pools of stable transfectants were selected by antibiotics or sorted by flow cytometry. Doxycycline (0.5 μg/mL) was used to induce shRNA while cumate (50 μg/mL) was used to induce shRNA-resistant cDNA expression.
Luciferase Reporter Assay. The luciferase plasmid LightSwitch 3′UTR Reporter, containing the Nanog 3′UTR or random negative control R01_3′UTR (Switchgear Genomics) was co-transfected with the m6A writer complex and ERK-activated kinase into HeLa cells for two days. Luciferase expression was measured using the Luciferase Assay System according to the commercial protocol (Promega). Nanog 3′UTR luciferase activity was normalized to cells transfected with R01_3′UTR.
Flow Cytometry. Flow cytometry analysis was conducted on BD LSR Fortessa and cell sorting was conducted on BD FACSAria Fusion. For alkaline phosphatase (Wiederschain et al.) staining, mESCs were incubated with fluorescent AP live stain (Sigma) for 30 min. For SSEA-1 expression, cells were disaggregated with trypsin, blocked with TruStain FcX (Biolegend) then incubated with anti-S SEA-1 (Biolegend) in cell staining buffer (Biolegend)
CRISPR Screen. The genome-wide CRISPR-Cas9 gene knockdown screen was accomplished using GeCKOv2 gene knockout library following published protocol (Joung et al., 2017). Briefly, the GECKOV2 library was amplified in Endura electrocompetent cells (Lucigen) then co-transfected with pMD2.G and psPAX2 into 293TN cells to produce a lentiviral library. HeLa-circGFP cells were infected at 0.3 MOI for 3 days, then selected with 2 μg/ml puromycin for 1 week before flow cytometry sorting. Genomes of harvested cells were extracted by Quick-gDNA MidiPrep (Zymo). sgRNA after PCR amplification were sent to the University of Chicago Genomics Facility to be sequenced on Illumina HiSeq 4000 in single-end read mode. RIGER was used to analyze the sequencing results. To obtain the ranked difference plot, sgRNAs were ranked according to the difference between number of reads in low and high GFP populations. The top 1% of the sgRNAs that ranked with the greatest difference were selected for gene ontology enrichment analysis.
Immunoprecipitation, immunoblotting, and in vitro kinase assay. Immunoprecipitation (IP) and immunoblotting (IB) were performed as previously described (Sun et al., 2016). In brief, protein samples were isolated from respective cells by lysis in RIPA buffer (1% Triton X-100, 150 mM NaCl, 20 mM Na2PO4, pH 7.4) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Subsequently, a BCA assay (Thermo Scientific) was used to determine the protein concentrations. For IP, indicated antibody and protein A/G magnetic beads (Thermo Scientific) were incubated with lysate at 4° C. overnight followed by washing and elution with sample buffer. Equal amounts of protein were separated by SDS-PAGE followed by wet transfer to PVDF membranes. Blots were blocked with 5% non-fat milk or BSA and incubated with respective primary antibody at 4° C. overnight. Primary antibodies were detected by HRP-linked secondary antibodies (Cell Signaling) together with SuperSignal West Pico Plus chemiluminescent substrate (Thermo Scientific) and imaged in a FluorChem R system (ProteinSimple).
Phosphate-affinity gel electrophoresis was performed in gels containing 60 μM MnCl2, and 30 μM acrylamide-pendant Phos-tag ligand (AAL-107, Wako Chemicals). For in vitro ERK2 kinase assays, recombinant full-length human ERK2 expressed in E. coli cells with an N-terminal GST tag and activated by MEK1, and N-terminal GST-tagged human METTL3/METTL14 complex expressed in Sf9 insect cells were purchased from SignalChem. Active ERK2 was serially diluted in Kinase Dilution Buffer III (SignalChem) and incubated with METTL3/METTL14 at 30° C. for 15 min. The reaction was stopped by the addition of the sample buffer then analyzed by IB.
Confocal Microscopy. For confocal microscopy, cells after treatments were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with 5% bovine serum albumin, incubated with primary antibodies overnight at 4° C. followed by incubation with the appropriate secondary antibody tagged with Alexa 488 or Alexa 568 (Molecular Probes). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) before mounting. Confocal fluorescence images were captured using Olympus FV1000 confocal spectral microscope.
Mass Spectrometry. To identify phosphorylation sites of METTL3, METTL3 precipitated from 293T cells co-transfected with myc-METTL3 and B-Raf V600E was analyzed by SDS-PAGE. The protein band corresponding to METTL3 was excised and subjected to in-gel digestion with tryspin and chymotrypsin. Samples were analyzed by Ultimate Capillary LC system (Dionex) directly coupled to LTQ Orbitrap Mass Analyzer (Thermo Scientific) using the TopTenTM method. The data were searched on MASCOT (MassMatrix) against the human Swiss-Prot database. All the identified phospho-peptides were further confirmed by manually checking the results.
RNA Extraction and Real-Time qPCR. Total RNA was isolated using TRIzol (Invitrogen), and 200 ng of RNA was reversed transcribed into cDNA using PrimeScript RT Reagent Kit (Takara). Real-time qPCR was performed using the FastStart Essential DNA Green Master (Roche). HPRT1 was used as an internal control for normalization. Primers used in this study are listed below. For measuring RNA stability, cells were treated with 5 μg/ml actinomycin D and harvested at 0, 3, and 6 hr to determine the half-life of target mRNAs.
LC-MS/MS quantification of m6A in poly(A) RNA. mRNA was extracted from the total RNA using 2 rounds of the Dynabeads mRNA purification kit. 100 ng of mRNA was digested by nuclease P1 (1U) in 20 μl of buffer containing 20 mM NH4OAc (pH=5.3) at 42° C. for 2 h, followed by dephosphorylation with the addition of FastAP Thermosensitive alkaline phosphatase (1U) and FastAP buffer at 37° C. for 2 h. The sample was then diluted to 50 μl and filtered (0.22 μm pore size, 4 mm diameter, Millipore). 5 μl of the solution was separated by reverse phase ultra-performance liquid chromatography on a C18 column, followed by online mass spectrometry detection using an Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified by using the nucleoside-to-base ion mass transitions of 282 to 150 (m6A) and 268 to 136 (A). Quantification was carried out by comparison with a standard curve obtained from pure nucleoside. The ratio of m6A to A was calculated based on the calibrated concentrations (Liu et al., 2018).
m6A-IP and m6A-seq. m6A-IP was performed using the EpiMark N6-Methyladenosine enrichment kit (NEB). Full length purified mRNA was used in m6A-IP-qPCR. For m6A-seq, mRNA was adjusted to 15 ng/μl in 100 μl and fragmented using a BioRuptor ultrasonicator (Diagenode) with 30 s on/off for 30 cycles. Input and RNA eluted from m6A-IP were used to prepare libraries with TruSeq Stranded mRNA Library Prep Kit (Illumina). Sequencing was carried out at the University of Chicago Genomics Facility on Illumina HiSeq 4000 in single-end read mode with 50 bp reads per read. Reads were aligned to the mycoplasma genome to assess contamination, followed by alignment to mouse genome version 10 (mm10) with HISAT2 v2.1.0 (Kim et al., 2015) with parameter −k 1.
The input library of m6A sequencing is used for comparing gene expression levels. DESeq2 (Love et al., 2014) was applied for differential expression between R-WT and R-3A2A mESCs with FDR<0.05 cutoff. m6A-seq data were analyzed as described before (De Jesus et al., 2019). m6A peak calling was performed using exomePeak R/Bioconductor package v 3.7 (Meng et al., 2013). Significant peaks with false discovery rate less than 0.05 were annotated to the RefSeq database (mm10). Homer v4.9.1 (Heinz et al., 2010) was used to search for the enriched motif in the m6A peak region where random peaks of 200 bp were used as background sequences for motif discovery. m6A peak distribution on the metagene was plotted by the R package Guitar (Cui et al., 2016).
Differential analysis of m6A methylation of patient samples was performed using the R package RADAR and MeRIPtools (Z. Z., M. Eckert, A. Zhu, A. Chryplewicz, D. F. D. J., D. Ren, R. N. K., E. Lengyel, C. H., and M. C.; unpublished observations). To summarize and visualize the m6A methylome data, principal component analysis (PCA) was performed using singular value decomposition approach implemented in R function (prcomp) on the logtransformed m6A-IP data. Pathway and gene ontology enrichment analysis were performed using WebGestalt (Liao et al., 2019) with default settings. Pathway enrichment terms were determined using WikiPathway and KEGG terms.
Cell Proliferation Assay. Cells were seeded in 96-well plates. The cell proliferation was assessed by SRB assay (Vichai and Kirtikara, 2006) at various time points. Briefly, cells after treatments were fixed with 10% TCA then stained with 0.05% SRB. After wash, bound SRB was solubilized with 10 mM Trizma base and measured at 515 nm.
Quantification of Histone Modifications. Histones were prepared from fresh cell pellets using Total Histone Extraction Kit (Epigentek). The efficiency of histone extraction was controlled by Coomassie blue staining and LB with anti-H3 antibody. Histone posttranslational modifications were quantified using the Histone H3 Modification Multiplex Assay Kit (Epigentek) following commercial protocol. Each histogram corresponds to the mean of 2 independent experiments and each measure was obtained using a pool of 100 ng of total histones from 2 independent extractions.
Statistical Analysis. Each experiment was performed at least three times, and representative data are shown. Data in the bar graphs are given as the mean±SEM. Means were checked for statistical difference using Student's t test, and p-values<0.05 were considered significant (*p<0.05, **p<0.01, ***p<0.001). For survival analysis, Kalpan-Meier analysis and a log rank test were applied.
Data Availability. The CRISPR screening and m6A-seq data generated during this study are available at GSE138776. The human data for the skin cutaneous melanoma (SKCM), was derived from the Cancer Genome Atlas (TCGA).
This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62/984,679 filed Mar. 3, 2020, and U.S. Provisional Patent Application No. 63/040,080 filed Jun. 17, 2020, both of which are incorporated herein by reference in their entirety and for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/020653 | 3/3/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62984679 | Mar 2020 | US | |
| 63040080 | Jun 2020 | US |
