RNA-based inhibitors of tRNA-modifying enzymes

Abstract

The present disclosure provides RNA molecules comprising at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme, wherein the formation of the covalent bond inhibits the tRNA modifying enzyme, and compositions comprising the RNA molecules. The present disclosure further provides methods for killing cells, particularly cancer cells, and methods for treating or preventing cancer in a subject. TIFF2026508359000008.tif91170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 487,980, filed March 02, 2023, which is incorporated by reference herein in its entirety.

[0002] Statement of Rights to Inventions Made Under Federally Sponsored Research This invention was made with government support under grants F31CA254339 and 5R21CA246118-02 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention.

[0003] Reference to the Electronic Sequence Listing The XML file named "047162-7443WO1 Seq Listing.xml", created on March 1, 2024, containing 56.7 kilobytes, is incorporated herein by reference in its entirety. [Background technology]

[0004] background Many known anticancer chemotherapeutics have significant side effects due to off-target inhibition of cellular pathways, such as DNA replication. For example, 5-fluorouracil (5-FU) is a known anticancer drug that can be misincorporated into the DNA of drug-treated cells, and genomic 5-FU accumulation is thought to correlate with 5-FU cytotoxicity in mammalian cells. Therefore, these compounds have significant off-target effects.

[0005] There is a need in the art for compositions and methods that are effective against cancer and / or have reduced off-target effects. The present invention addresses this need. Summary of the Invention

[0006] overview In some aspects, the present invention relates to an RNA molecule comprising at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme, wherein the formation of the covalent bond inhibits the tRNA modifying enzyme.

[0007] In some aspects, the present invention relates to a composition comprising the RNA molecule, hi some embodiments, the composition is a pharmaceutical composition.

[0008] In some aspects, the present invention relates to a method for killing a cell, in some embodiments, the method comprises contacting the RNA molecule with a tRNA modifying enzyme in the cell, in some embodiments, the formation of a covalent bond inhibits the tRNA modifying enzyme, thereby killing the cell.

[0009] In some aspects, the present invention relates to a method for treating cancer in a subject in need thereof. In some embodiments, the method comprises administering to the subject an effective amount of a pharmaceutical composition. In some embodiments, the RNA molecule contacts a tRNA-modifying enzyme in cancer cells of the cancer, and the formation of a covalent bond inhibits the tRNA-modifying enzyme, thereby killing the cancer cells.

[0010] In some aspects, the present invention relates to the following non-limiting embodiments.

[0011] RNA molecule In some aspects, the present invention relates to RNA molecules.

[0012] In some embodiments, the RNA molecule comprises at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme.

[0013] In some embodiments, the formation of the covalent bond inhibits a tRNA modifying enzyme.

[0014] In some embodiments, the RNA molecule is a tRNA molecule.

[0015] In some embodiments, at least one nucleotide comprises an unnatural base.

[0016] In some embodiments, at least one nucleotide is 5-halouridine (5-haloU), 5-halocytidine (5-haloC), 5-aza-cytidine (5-azaC), 8-haloadenosine (8-haloA), 8-azanebularine, 8-aza-adenosine (8-azaA), or 8-haloguanosine (8-haloG).

[0017] In some embodiments, the tRNA modifying enzyme is dihydrouridine synthase (DUS) or pseudouridine synthase (PUS).

[0018] In some embodiments, the tRNA modifying enzyme is dihydrouridine synthase 1 (DUS1), dihydrouridine synthase 2 (DUS2), dihydrouridine synthase 3 (DUS3), dihydrouridine synthase 4 (DUS4), dihydrouridine synthase 1-like (DUS1L), dihydrouridine synthase 3-like (DUS3L), dihydrouridine synthase 4-like (DUS4L), pseudouridine synthase 1 (PUS1), pseudouridine synthase-like 1 (PUSL1), pseudouridine synthase 3 (PUS3), TruB pseudouridine synthase family member 1 (TRUB1), TruB pseudouridine synthase family member 2 (TRUB2), dyskerin pseudouridine synthase 1 (DKC1), pseudouridine synthase 7 (PUS7), pseudouridine synthase 1 (PUS1L ... Uridine synthase 7-like (PUS7L), RNA pseudouridylate synthase domain-containing 1 (RPUSD1), RNA pseudouridylate synthase domain-containing 2 (RPUSD2), RNA pseudouridylate synthase domain-containing 4 (RPUSD4), pseudouridine synthase 10 (PUS10), tRNA methyltransferase 2 homolog A (TRMT2A), tRNA methyltransferase 2 homolog B (TRMT2B), NOP2 / SunRNA methyltransferase 2 (NSUN2), NOP2 / SunRNA methyltransferase 3 (NSUN3), NOP2 / SunRNA methyltransferase 6 (NSUN6), DNA methyltransferase 2 (DNMT2), Methyltransferase-Like Protein 1 (Methyltransferase-Like Protein 1) Protein 1 (METTL1), WD repeat domain 4 (WDR4), adenosine deaminase TRNA-specific 1 (ADAT1), adenosine deaminase TRNA-specific 2 (ADAT2), adenosine deaminase TRNA-specific 2 (ADAT3), ISCU, or a combination thereof.

[0019] In some embodiments, the unnatural base is 5-halo U and the tRNA modifying enzyme is DUS2, DUS1L, DUS3L, DUS4L, ISCU, PUS1, PUS3, PUS7, PUS10, PUSL1, PUS7L, RPUSD1, RPUSD2, RPUSD4, TRMT2A, TRMT2B, TRUB1, TRUB2, or a combination thereof.

[0020] In some embodiments, the nucleotide comprising an unnatural base is 5-azaC and the tRNA modifying enzyme is DNMT2, NSUN2, NSUN3, NSUN6, or a combination thereof.

[0021] In some embodiments, the nucleotide comprising an unnatural base is an 8-halo-G and the tRNA modifying enzyme is METTL1, WDR4, or a combination thereof.

[0022] In some embodiments, the nucleotide comprising an unnatural base is 8-AzaA and the tRNA modifying enzyme is ADAT1, ADAT2, ADAT3, or a combination thereof.

[0023] In some embodiments, at least one nucleotide is at a position that corresponds to the natural position of a natural nucleotide of the natural tRNA that is modified by the tRNA modifying enzyme.

[0024] In some embodiments, the RNA molecule is a tRNA molecule and the non-naturally modified base is a 5-halouracil within the D-loop, t-psi-c loop, anticodon loop, RNA stem, and other portions of the tRNA molecule.

[0025] In some embodiments, the RNA molecule comprises the sequence of any one of SEQ ID NOs: 1-53 or comprises at least about 80% sequence identity to the sequence of any one of SEQ ID NOs: 1-53.

[0026] In some embodiments, the RNA molecule is an isolated tRNA molecule.

[0027] In some embodiments, the RNA molecule comprises two or more unnatural bases.

[0028] In some embodiments, the two or more unnatural bases inhibit two or more different tRNA modifying enzymes.

[0029] composition In some aspects, the present invention relates to a composition.

[0030] In some embodiments, the composition comprises an RNA molecule described herein.

[0031] In some aspects, the composition further comprises a pharmaceutically acceptable carrier.

[0032] In some embodiments, the composition is a pharmaceutical composition.

[0033] Methods for killing cells In some aspects, the present invention relates to a method for killing a cell.

[0034] In some embodiments, the methods comprise contacting an RNA molecule herein with a tRNA modifying enzyme in a cell.

[0035] In some embodiments, the formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cell.

[0036] In some aspects, the cell is a brain cancer cell, a gastrointestinal cancer cell, a kidney cancer cell, a liver cancer cell, or a lung cancer cell.

[0037] In some embodiments, the cells are bladder cancer cells, breast cancer cells, cervical cancer cells, cholangiocarcinoma cells, colon cancer cells, esophageal cancer cells, head / neck cancer cells, renal clear cell carcinoma cells, papillary renal carcinoma cells, liver cancer cells, non-small cell lung carcinoma cells, small cell lung carcinoma cells, prostate cancer cells, rectal cancer cells, sarcoma cancer cells, gastric cancer cells, uterine cancer cells, liquid tumor (e.g., acute myeloid leukemia / AML, etc.) cells, etc.

[0038] In some aspects, the cells are cancer cells in culture.

[0039] Methods for treating cancer In some aspects, the present invention relates to methods for treating cancer in a subject in need thereof.

[0040] In some aspects, the methods comprise administering to a subject an effective amount of a pharmaceutical composition herein.

[0041] In some embodiments, the RNA molecule contacts a tRNA modifying enzyme in a cancer cell of the cancer.

[0042] In some embodiments, the formation of the covalent bond inhibits tRNA-modifying enzymes, thereby killing the cancer cells.

[0043] In some aspects, the cancer is lung cancer, brain cancer, gastrointestinal cancer, kidney cancer, liver cancer, or a combination thereof.

[0044] In some embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, bile duct cancer, colon cancer, esophageal cancer, head / neck cancer, kidney clear cancer, papillary renal cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, prostate cancer, rectal cancer, sarcoma cancer, gastric cancer, uterine cancer, liquid tumors (e.g., acute myeloid leukemia / AML, etc.), etc.

[0045] In some aspects, the method further comprises administering chemotherapy to the subject. [Brief explanation of the drawings]

[0046] [Figure 1A] 1A-1B illustrate non-limiting examples of tRNA-modifying enzymes that are upregulated in cancer. Figure 1A: Dihydrouridine synthase (DUS) is upregulated in cancer. Figure 1B: Pseudouridine synthase (PUS) is upregulated in cancer. [Figure 1B] See legend to Figure 1A. [Figure 2]FIG. 2 illustrates that 5-halouracil forms a covalent bond with DUS, according to some embodiments. [Figure 3] FIG. 3 illustrates non-limiting examples of tRNA molecules of the invention covalently linked to a tRNA modifying enzyme, according to some embodiments. [Figure 4] FIG. 4 depicts a Western blot confirming that non-limiting examples of RNA molecules described herein can form covalent bonds with DUS2, a non-limiting tRNA-modifying enzyme, according to some embodiments. [Figure 5] FIG. 5 is a graph showing that the RNA molecules of FIG. 4 were able to kill cells of the lung adenocarcinoma cell line PC9, according to some embodiments. [Figure 6A] Figures 6A-6I illustrate, according to some embodiments, that DUS2 is overexpressed in lung cancer and that loss of DUS2 sensitizes cells to ferroptosis. Figure 6A: Schematic of key regulators and chemical effectors of ferroptosis. Figure 6B: High DUS2 mRNA expression correlates with resistance to chemical ferroptosis inducers in a panel of 860 cancer cell lines. Figure 6C: The DUS enzyme converts uridine to dihydrouridine. Figure 6D: DUS2 RNA levels are significantly higher in lung adenocarcinoma (LUAD) tumor samples compared to normal tissues (two-tailed Mann-Whitney U test, CysGCA p<0.0001). Figure 6E: High DUS2 expression predicts worse outcome in NSCLC patients. Figure 6F: DUS2 protein expression is absent in clonal A549 DUS2 KO cells. Figure 6G: Increased cell death in DUS2 KO compared to WT A549 after treatment with 2 μM RSL-3 (Annexin V+ / PI+ cells). Figure 6H: Elevated lipid ROS in DUS2 KO compared to WT A549 after treatment with 2 μM RSL3 (C11-BODIPY staining). Figure 6I: Quantification of Figure 6H (two-tailed t-test, *p<0.0001). [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E]See legend to Figure 6A. [Figure 6F] See legend to Figure 6A. [Figure 6G] See legend to Figure 6A. [Figure 6H] See legend to Figure 6A. [Figure 6I] See legend to Figure 6A. [Figure 7A] Figures 7A-7F illustrate that DUS2 is required to maintain the levels of a specific tRNA, tRNACysGCA, according to some embodiments. Figure 7A: DUS2 targets position U20 of the tRNA. Figure 7B: Quantification of tRNA charging levels in DUS2 WT and KO cells. tRNA charging was unaffected by DUS2 loss, whereas Gln starvation reduces tRNAGln charging levels by >50%. Figure 7C: Changes in tRNA levels in DUS2 KO cells; black dots (FDR<0.05), gray (FDR>0.05), and red (tRNACysGCA and FDR<0.05). Figure 7D: In DUS2 KO, an exemplary isodecoder, tRNACysGCA10-1, shows approximately 50% less expression. Figure 7E: Analysis of tRNA levels in TCGA LUAD data. The tRNACysGCA level in tumors was significantly higher than that in normal tissues, but the tRNAGlnCTG level was not (two-tailed Mann-Whitney U test, CysGCA p<.0001). [Figure 7B] See the legend to Figure 7A. [Figure 7C] See the legend to Figure 7A. [Figure 7D] See the legend to Figure 7A. [Figure 7E] See the legend to Figure 7A. [Figure 7F] See the legend to Figure 7A. [Figure 8A]Figures 8A-8I illustrate that, according to some embodiments, loss of DUS2 impairs the translation of cysteine-rich proteins, including metallothionein, leading to ferroptosis susceptibility. Figure 8A: Cysteine ​​translation reporter. Figure 8B: DUS2 KO impairs translation of TGT and TGC cysteine ​​codons (two-tailed t-test, *p<0.03). Figure 8C: Transfection of DUS2 KO cells with tRNACysGCA rescues TGT translation (two-tailed t-test, *p<0.0001). Figure 8D: Proteins with high Cys content (>5%) are reduced in DUS2 KO cells. Cumulative distribution of changes in protein abundance (log2 fold change, Kolmogorov-Smirnov test, p<0.05). Figure 8E: Metallothionein translation reporter. Figure 8F: Metallothioneins (MT1A and MT1G) are translated less in DUS2 KO. Figure 8G: Increased cell death (Annexin V+ / PI+ cells) in DUS2 KO compared to WT A549 after addition of 62.5 μM ZnCl2. Figure 8H: DUS2 KO cells have a smaller level of GSH reduction (two-tailed t-test, *p<0.004). Figure 8I: Model for the anti-ferroptosis function of DUS2. [Figure 8B] See the legend to Figure 8A. [Figure 8C] See the legend to Figure 8A. [Figure 8D] See the legend to Figure 8A. [Figure 8E] See the legend to Figure 8A. [Figure 8F] See the legend to Figure 8A. [Figure 8G] See the legend to Figure 8A. [Figure 8H] See the legend to Figure 8A. [Figure 8I] See the legend to Figure 8A. [Figure 9A]Figures 9A-9G illustrate that combining DUS2 loss and ferroptosis induction in a mouse xenograft NSCLC model extends lifespan, according to some embodiments. Figure 9A: A549 tumors grow faster than DUS2 KO tumors. Figure 9B: Example tumors derived from A549 and DUS2 KO-2. Figure 9C: By qRT-PCR, DUS2 KO tumors express higher PTGS2, a marker of ferroptosis, than A549 cells (ANOVA, *p<0.002). Figure 9D: Dosing scheme for xenograft experiments. Figure 9E: Oral JKE-1674 treatment induces PTGS2 mRNA in mouse lungs (ANOVA, *p<0.03). Figure 9F: JKE-1674 treatment increases PTGS2 mRNA expression in DUS2 KO tumors (ANOVA, *p<0.02). Figure 9G: DUS2 KO xenograft tumor-bearing mice survive longer than A549 tumor-bearing mice upon JKE-1674 treatment (Mantel-Cox test *p<0.03). [Figure 9B] See the legend to Figure 9A. [Figure 9C] See the legend to Figure 9A. [Figure 9D] See the legend to Figure 9A. [Figure 9E] See the legend to Figure 9A. [Figure 9F] See the legend to Figure 9A. [Figure 9G] See the legend to Figure 9A. [Figure 10A] Figures 10A-10E show that the percentage of dead (Annexin V / PI) DUS2 KO cells was reduced by pretreatment with a DUS2 expression plasmid, ferrostatin-1, or Trolox, but not ZVAD-FMK, when treated with 2 μM RSL-3. Figure 10D: When treated with 200 nM ML162, DUS2 KO cells have elevated lipid ROS levels as measured by C11-BODIPY staining. Figure 10E: When treated with 2 μM RSL-3, DUS2 KO cells have elevated cellular ROS levels as measured by H2DCFDA staining. [Figure 10B] See the legend to Figure 10A. [Figure 10C] See the legend to Figure 10A. [Figure 10D] See the legend to Figure 10A. [Figure 10E] See the legend to Figure 10A. [Figure 11] FIG. 11 illustrates that the total pool of tRNACysGCA was reduced by approximately 40% in the DUS2 KO clone in aggregates. [Figure 12A] Figure 12A illustrates that total protein synthesis was intact in DUS2 KO cells as measured by 35S-Met incorporation (two-tailed t-test, *p=0.67). Figure 12B: Consistent with RQC, mRNAs encoding cysteine-rich proteins were reduced in DUS2 KO cells. Cumulative distribution of changes in mRNA abundance (log2 fold change, KS test, p<0.0001). Figure 12C: Depletion of the RQC factor GIGYF2 (siGIGYF2) in DUS2 KO cells rescues MT1A mRNA levels compared to the non-targeting control (siNT) (two-tailed t-test, *p<0.04). [Figure 12B] See the legend to Figure 12A. [Figure 12C] See the legend to Figure 12A. [Figure 13] Figures 13A-13B illustrate that mouse weight generally increased over time with vehicle and JKE-1674. Figure 13B: Median survival of mice given JKE-1674 was shorter than that of mice given vehicle. [Figure 14] Figures 14A-14B demonstrate that, according to some embodiments, CLB-001, a non-limiting example of an RNA-based DUS inhibitor herein, was able to kill cancer cells at nanomolar IC levels. A hepatocellular carcinoma cell line, HepG2 (Figure 14A), and a non-small cell lung cancer cell line, A549 (Figure 14B), were subjected to various concentrations of CLB-001. Cell viability was plotted against CLB-001 concentration. [Figure 15]Figure 15 demonstrates that, according to some embodiments, CLB-001, a non-limiting example of an RNA-based DUS inhibitor herein, does not kill non-cancer cells. A non-transformed hepatocyte cell line, AML12, was subjected to various concentrations of CLB-001. Cell viability was plotted against CLB-001 concentration. [Figure 16] Figure 16 demonstrates that, according to some embodiments, CLB-001, a non-limiting example of an RNA-based DUS inhibitor herein, is significantly more potent than 5-FU. HepG2 cells were subjected to various concentrations of CLB-001. Cell viability was plotted against CLB-001 concentration. While the active moiety of CLB-001 is 5-FU, 5-FU was incorporated into a tRNACys ​​molecule, dramatically increasing the drug's potency, making CLB-001 over 7,000 times more potent than 5FU in killing HepG2 cancer cells. [Figure 17A] 17A-17C demonstrate that tRNA-modifying enzymes are upregulated in hepatocellular carcinoma, according to some embodiments. The mRNA levels of various tRNA-modifying enzymes from hepatocellular carcinoma (HCC) tumor tissues were compared with the mRNA levels of various tRNA-modifying enzymes from normal liver tissues. tRNA-modifying enzymes were found to be consistently overexpressed in HCC tumors. [Figure 17B] See the legend to Figure 17A. [Figure 17C] See the legend to Figure 17A. [Figure 18A] 18A-18G demonstrate that high expression of tRNA-modifying enzymes predicts poor outcome in hepatocellular carcinoma (HCC), according to some embodiments. In each panel of the figure, HCC patients are grouped according to tRNA-modifying enzyme expression levels in tumor tissue, and patient survival percentages are plotted over time according to the grouping. [Figure 18B] See the description of Figure 18A. [Figure 18C] See the description of Figure 18A. [Figure 18D] See the description of Figure 18A. [Figure 18E] See the description of Figure 18A. [Figure 18F] See the description of Figure 18A. [Figure 18G] See the description of Figure 18A. [Figure 19] Figure 19 demonstrates that, according to some embodiments, CLB-001, a non-limiting example of an RNA-based DUS inhibitor and PUS inhibitor herein, can kill cancer cells at nanomolar IC50 levels. Hepatocellular carcinoma cell lines, SNU-387, HepG2, and PLC / PRF / 5, and non-small cell lung cancer cell line, A549, were subjected to various concentrations of CLB-001. Cell viability was plotted against CLB-001 concentration. DETAILED DESCRIPTION OF THE INVENTION

[0047] Detailed Description Without being limited by theory, the present invention is based in part on the discovery that substrate mimetic transfer RNAs (tRNAs) incorporating unnatural nucleotides at specific positions can be used to covalently capture and thereby inhibit tRNA-modifying enzymes (e.g., tRNA-modifying enzymes that promote cancer progression and / or metastasis).

[0048] definition 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 to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice and / or testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used depending on how it is defined, and the definitions are provided here.

[0049] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0050] It should also be understood that the methods described in this disclosure are not limited to the particular methods and experimental conditions disclosed herein, and as such, methods and conditions may vary.

[0051] Furthermore, unless otherwise specified, the experiments described herein use conventional molecular techniques, cell biological techniques and immunological techniques within the scope of the art.Such techniques are well known to skilled workers and are fully described in literature.For example, see Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008), including all supplements, MR Green and J. Sambrook's Molecular Cloning: A Laboratory Manual (Fourth Edition), and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd Edition).

[0052] Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the event of latent ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise specified. The use of the term "including" and other variations such as "includes" and "included" is not limiting.

[0053] In general, the terminology used in the fields of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and, unless otherwise specified, as described in the various general and more specific references cited and discussed throughout the specification. Enzymatic reactions and purification techniques are performed as commonly accomplished in the art or according to manufacturer's specifications as described herein. The terminology used in analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry testing methods and techniques described herein, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0054] In order that this disclosure may be more readily understood, selected terms are defined below.

[0055] As used herein, the articles "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0056] As used herein, the term "about," when referring to a measurable value, e.g., an amount, a duration, etc., is meant to encompass variations of ±20%, or within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value, when such variations are suitable for performing the disclosed methods. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0057] The term "isolated," as used herein with respect to any molecule, for example, in an "isolated RNA molecule," refers to a molecule that has been isolated from other cellular components, and is meant to encompass both purified and recombinant molecules. Thus, the term "isolated RNA molecule" refers to an RNA molecule that is substantially free of cellular material, viral material, or culture medium when, for example, produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

[0058] As used herein, "identity" refers to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules, e.g., between two RNA molecules. When two polynucleotide sequences have the same nucleotide at the same position, for example, if a position in each of the two nucleic acid molecules is occupied by uracil, they are identical at that position. Identity, i.e., the degree to which two sequences have the same thing, e.g., nucleotide, at the same position in an alignment, is often expressed as a percentage. Because two polynucleotides may each contain similar sequences (i.e., a portion of the complete polynucleotide sequence) between the two polynucleotides and may also contain different sequences between the two polynucleotides, sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. Homology or identity can be determined by sequence alignment, for example, using programs known in the art such as BLAST, ALIGN, or CLUSTAL.

[0059] As used herein, the terms "inhibit" or "inhibiting" a tRNA modifying enzyme refer to any statistically significant reduction in the biological activity of the tRNA modifying enzyme, including complete blocking of the activity.

[0060] The terms "non-natural" and "non-naturally occurring" refer to not occurring in nature. For example, a "non-natural" or "non-naturally occurring" nucleic acid or nucleotide refers to a nucleic acid or nucleotide that does not occur in nature. For example, a non-naturally occurring nucleic acid may contain one or more non-natural bases, sugars, and / or intersubunit linkages, e.g., sugars, bases, and / or linkages that are modified or substituted relative to the bases, sugars, and / or linkages found in naturally occurring nucleic acid molecules. In some embodiments, a non-naturally occurring nucleic acid contains multiple types of modifications, including, but not limited to, sugar and base modifications, sugar and linkage modifications, base and linkage modifications, or base, sugar, and linkage modifications.

[0061] As used therein, a "subject" or "patient" may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals.

[0062] "Wild-type" means the non-mutated version of a gene, allele, genotype, nucleic acid, polypeptide, or phenotype, or a fragment of any of these, which may occur in nature or be produced recombinantly.

[0063] In this disclosure, terms such as "comprises," "comprising," "containing," and "having" may have the meaning ascribed to them under U.S. patent law and may refer to terms such as "includes," "including," and the like. Similarly, "consisting essentially of" or "consisting essentially of" has the meaning ascribed to it under U.S. patent law, and the term is open-ended, allowing for the presence of more than what is recited, as long as the basic or novel characteristics of the recited items are not altered by the presence of more than what is recited and prior art aspects are excluded. The use of the transitional phrase "consisting essentially of" means that the claim is to be interpreted to include the specified materials or steps recited in the claim, as well as those that do not materially affect the basic and novel characteristics of the invention. Thus, the term "consisting essentially of" when used in the claims of the present invention is not intended to be interpreted as equivalent to "comprising." In some embodiments, the term "consisting essentially of" refers to a composition in which the only active ingredient is the indicated active ingredient (e.g., the indicated RNA molecule). However, other ingredients may be included for stabilizing the formulation, preserving it, etc., but not directly involved in the therapeutic effect of the indicated active ingredient.

[0064] As used herein, the terms "treat," "treating," "ameliorating," "treatment," and the like refer to the alleviation or improvement of a disease or condition and / or one or more symptoms associated therewith. Although not excluded, it is understood that treating a disease or condition and / or one or more symptoms associated therewith does not require that the disease, condition, or symptoms associated therewith be completely ameliorated or eliminated. This means that clinical signs and / or symptoms associated with the disease or condition are alleviated as a result of the action taken. Signs or symptoms to be monitored are well known to skilled clinicians.

[0065] range: Throughout this application, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0066] The description of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0067] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0068] Molecules and Compositions In some aspects, the present invention provides RNA molecules that include at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme, wherein formation of the covalent bond inhibits the tRNA modifying enzyme.

[0069] RNA molecules can be prepared using methods known in the art, including, but not limited to, in vitro transcription and chemical synthesis. Nelissen, E. et al., Nucleic Acids Research, 40(13):e102 (2012); Milligan, JF et al., Nucleic Acids Research, 15:8783-8798 (1987); Marshall, WS et al., Curr. Opin. Chem. Biol., 8:222-229 (2004); Ponchon, L. et al., Nat. Protoc., 4:947-959 (2009); Ponchon, L. et al., Nat. Methods, 4:571-576 (2007); Ponchon, L. et al., Methods, 54:267-273 (2011), and U.S. Patent Application No. 2009 / 0298920, each of which is incorporated herein by reference in its entirety, describe methods for producing RNA.

[0070] In one embodiment, an RNA molecule includes any ribonucleic strand.

[0071] In another embodiment, the RNA molecule comprises a length of about 1,000 ribonucleotides, illustratively from about 5 to about 1,000, from about 10 to about 900, from about 20 to about 500, from about 30 to about 300, from about 40 to about 200, and from about 70 to about 100 ribonucleotides.

[0072] In other embodiments, the RNA molecule comprises from about 76 to about 96 ribonucleotides.

[0073] In other embodiments, the RNA molecule comprises from about 60 to about 86 ribonucleotides.

[0074] In some embodiments, the bond between at least the nucleotide and the tRNA modifying enzyme can be formed by any of a variety of direct or indirect covalent associations or attachments.

[0075] In one embodiment, at least one nucleotide can form a covalent bond with a tRNA modifying enzyme by forming a covalent bond directly or indirectly between a base of the at least one nucleotide and an amino acid residue of the tRNA modifying enzyme.

[0076] In other embodiments, the covalent attachment of at least one nucleotide to the tRNA modifying enzyme is irreversible.

[0077] In another embodiment, the covalent attachment of at least one nucleotide to the tRNA modifying enzyme inhibits the tRNA modifying enzyme. In one embodiment, inhibition comprises at least about a 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% decrease in tRNA modifying enzyme activity. In another embodiment, inhibition comprises a 100% decrease in tRNA modifying enzyme activity.

[0078] In some embodiments, the tRNA modifying enzyme is trapped and irreversibly inhibited by the formation of a covalent bond.

[0079] In some embodiments, at least one nucleotide is a non-natural nucleotide. In one embodiment, at least one nucleotide comprises a non-natural base.

[0080] In one embodiment, the RNA molecule comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 non-naturally occurring nucleotides, wherein independently, each nucleotide comprises an non-naturally occurring base.

[0081] Those skilled in the art will recognize that numerous "synthetic" non-natural nucleosides containing a variety of heterocyclic bases and / or a variety of sugar moieties (and sugar analogs) are available in the art, and that RNA molecules may contain one or more heterocyclic bases in addition to the five main base components of natural nucleic acids, so long as they meet the other criteria of the invention.

[0082] In some embodiments, heterocyclic bases include, but are not limited to, uracil-5-ylcytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo[2,3-d]methylpyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-5-yl, and 2-amino-4-oxopyrrolo[2,3-d]methylpyrimidin-3-yl groups, where purines are attached to the sugar moiety of the ISS through the 9 position, pyrimidines through the 1 position, pyrrolopyrimidines through the 7 position, and pyrazolopyrimidines through the 1 position.

[0083] In other embodiments, at least one nucleotide comprises at least one modified base. Examples of base modifications include, but are not limited to, uracils modified, preferably with halogen, at C-5 and / or C-6, including, but not limited to, fluorouracil, e.g., 5-fluorouracil (5-FU), bromouracil, e.g., 5-bromouracil, chlorouracil, e.g., 5-chlorouracil, and iodouracil, e.g., 5-iodouracil and hydroxyuracil. Other examples of base modifications include 8-azaadenosine (8-aza-Ad), 7-deazaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine Cytosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7deaza-8-azaguanosine, azacytosine, 5-bromocytosine, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, 5,6-dihydrocytosine, 5-iodocytosine, 5-nitrocytosine, 5-hydroxy-cytosine, 6-thio-guanine, and 4-thiouracil.

[0084] In one embodiment, the at least one nucleotide comprising an unnatural base is 5-halo (halo = F / Cl / Br / I) uridine, 5-halo (halo = F / Cl / Br / I) cytidine, 5-aza-cytidine, 8-halo (halo = F / Cl / Br / I) adenosine, 8-azanebularine, 8-aza-adenosine, or 8-halo (halo = F / Cl / Br / I) guanosine.

[0085] In some embodiments, the amino acid residue of the tRNA modifying enzyme is a conserved catalytic amino acid residue.

[0086] In other embodiments, the amino acid residue of the tRNA modifying enzyme is a reactive amino acid residue.

[0087] The amino acid residues of the tRNA modifying enzymes are reactive nucleophilic amino acid residues.

[0088] In some embodiments, the reactive nucleophilic amino acid residue is a reactive cysteine ​​residue, and covalent bond formation occurs via nucleophilic attack of at least one nucleotide by the reactive cysteine ​​residue.

[0089] In another embodiment, at least one nucleotide of the RNA molecule is a 5-halopyrmidine, and at least one nucleotide is capable of forming a covalent bond with a conserved catalytic cysteine ​​residue of a tRNA-modifying enzyme, thereby covalently linking the RNA molecule and the tRNA-modifying enzyme.

[0090] Without being limited by theory, in some embodiments, crosslinking begins with the reduction (e.g., enzymatic reduction) of 5-halouridine to 5-halodihydrouridine, followed by nucleophilic attack of the conserved catalytic cysteine ​​of the tRNA-modifying enzyme at the C5 position by the halide, which acts as a leaving group. Dai, W. et al., Nat Chem Biol., 17(11):1178-1187 (2021) describes activity-based RNA-modifying enzyme probing and is incorporated herein by reference in its entirety.

[0091] In one embodiment, the RNA molecule is a tRNA molecule.

[0092] The general characteristics of natural tRNAs are well known to those skilled in the art. Sprinzl, M. et al., Nucleic Acids Research, 26(1):148-153 (1998), which is incorporated herein by reference in its entirety, describes the editing of tRNA genes and the sequence of tRNA genes.

[0093] In some embodiments, a tRNA molecule of the invention is a single ribonucleotide strand that can fold into a characteristic, so-called cloverleaf secondary structure.

[0094] In some embodiments, the secondary structure is: (i) an acceptor stem that is composed of the first seven ribonucleotides at the 5' end of the ribonucleotide chain and the seven ribonucleotides preceding the last four ribonucleotides at the 3' end of the ribonucleotide chain, thus forming a double-stranded structure containing approximately six or seven pairs of ribonucleotides, such that the ribonucleotides including the first ribonucleotide at the 5' end of the ribonucleotide chain are not paired with the ribonucleotides preceding the last four ribonucleotides at the 3' end of the ribonucleotide chain; (ii) a D arm that contains four pairs of ribonucleotides and a D loop that contains approximately 8 to 10 ribonucleotides, formed by folding back a portion of the ribonucleotide chain following the first seven ribonucleotides at the 5' end of the ribonucleotide chain; (iii) an anticodon stem that contains five pairs of ribonucleotides and an anticodon loop that contains approximately seven ribonucleotides (anticodon stem-loop), formed by folding back a portion of the ribonucleotide chain following the D arm and the D loop; (iv) (v) a variable loop comprising about 4 to about 21 ribonucleotides and formed by the anticodon stem and a portion of the ribonucleotide chain following the anticodon loop; (v) a T-arm comprising about 5 pairs of ribonucleotides and a T-loop comprising about 8 ribonucleotides, formed by folding back a portion of the ribonucleotide chain following the variable loop and preceding the 3'-terminal ribonucleotide of the ribonucleotide chain comprising the acceptor stem.

[0095] In some embodiments, the tRNA-modifying enzyme is a dihydrouridine synthase (DUS) or a pseudouridine synthase (PUS). Both DUS and PUS are known to contain highly conserved cysteine ​​or aspartic acid residues in their respective active sites.

[0096] In some embodiments, the tRNA modifying enzyme is dihydrouridine synthase 1 (DUS1), dihydrouridine synthase 2 (DUS2), dihydrouridine synthase 3 (DUS3), dihydrouridine synthase 4 (DUS4), dihydrouridine synthase 1-like (DUS1L), dihydrouridine synthase 3-like (DUS3L), dihydrouridine synthase 4-like (DUS4L), pseudouridine synthase 1 (PUS1), pseudouridine synthase-like 1 (PUSL1), pseudouridine synthase 3 (PUS3), TruB pseudouridine synthase family member 1 (TRUB1), TruB pseudouridine synthase family member 1 (TRUB2), TruB pseudouridine synthase family member 1 (TRUB3), TruB pseudouridine synthase family member 1 (TRUB4), TruB pseudouridine synthase family member 1 (TRUB5), TruB pseudouridine synthase family member 1 (TRUB6), TruB pseudouridine synthase family member 1 (TRUB7), TruB pseudouridine synthase family member 1 (TRUB8), TruB pseudouridine synthase family member 1 (TRUB9), TruB pseudouridine synthase family member 1 (TRUB11), TruB pseudouridine synthase family member 1 (TRUB12), TruB pseudouridine synthase family member 1 (TRUB13), TruB pseudouridine synthase family member 1 (TRUB14), TruB pseudouridine synthase family member 1 (TRUB15), TruB pseudouridine synthase family member 1 (TRUB16), TruB pseudouridine synthase family member 1 (TRUB17), TruB pseudouridine synthase family member 1 (TRUB18), TruB pseudouridine synthase family member 1 (TRUB19), TruB pseudouridine synthase family member 1 (TRUB19), TruB pseudouridine synthase family member 1 (TRUB19), tRNA methyltransferase family member 2 (TRUB2), dyskerin pseudouridine synthase 1 (DKC1), pseudouridine synthase 7 (PUS7), pseudouridine synthase 7-like (PUS7L), RNA pseudouridylate synthase domain-containing 1 (RPUSD1), RNA pseudouridylate synthase domain-containing 2 (RPUSD2), RNA pseudouridylate synthase domain-containing 4 (RPUSD4), pseudouridine synthase 10 (PUS10), tRNA methyltransferase 2 homolog A (TRMT2A), tRNA methyltransferase 2 homolog B (TRMT2B), NOP2 / Sun RNA methyltransferase 2 (NSUN2), NOP2 / Sun RNA methyltransferase 3 (NSUN3), NOP2 / Sun RNA methyltransferase 6 (NSUN6), DNA methyltransferase 2 (DNMT2), methyltransferase-like protein 1 (METTL1), WD repeat domain 4 (WDR4), adenosine deaminase TRNA-specific 1 (ADAT1), adenosine deaminase TRNA-specific 2 (ADAT2), adenosine deaminase TRNA-specific 2 (ADAT3), or ISCU.

[0097] In some embodiments, the unnatural base in the RNA molecule is selected based on the target tRNA modifying enzyme that the RNA molecule inhibits. A non-limiting list of nucleotides and tRNA modifying enzymes containing unnatural bases is provided in Table 1 below.

[0098] Table 1. Examples of enzymes and nucleotides containing unnatural bases TIFF2026508359000002.tif20490

[0099] In some embodiments, the unnatural base is 5-halo U and the tRNA modifying enzyme is DUS2, DUS1L, DUS3L, DUS4L, ISCU, PUS1, PUS3, PUS7, PUS10, PUSL1, PUS7L, RPUSD1, RPUSD2, RPUSD4, TRMT2A, TRMT2B, TRUB1, TRUB2, or a combination thereof.

[0100] Base modifications of naturally occurring tRNA molecules by tRNA modifying enzymes in humans and other species have been extensively characterized, and there is a large amount of information available regarding the naturally occurring tRNA molecules modified by these enzymes, as well as the specific naturally occurring nucleotides of naturally occurring tRNA molecules modified by these enzymes. Thus, in some embodiments, the tRNA molecules of the invention comprise the sequence of a naturally occurring tRNA molecule in which at least one nucleotide is a non-naturally occurring nucleotide located at a nucleotide position in the tRNA molecule that corresponds to the natural position, or is a non-naturally occurring nucleotide located no more than 5, 4, 3, 2, or 1 nucleotide position away from the natural position in the naturally occurring tRNA molecule.

[0101] Because tRNA modifying enzymes, such as those described herein, are known to modify bases located at virtually any position in a tRNA molecule, the location of the unnatural base is not limited. In some embodiments, the location of the unnatural base is determined based on the enzyme to be inhibited, the sequence of the parent natural tRNA, and the unnatural base. For example, DUS enzymes can modify uridines in the D-loop of tRNA (as well as uridines outside the D-loop, such as the tRNA stem) and can be inhibited by 5-halouridine. Thus, in some embodiments, inhibitors specific to DUS enzymes can be designed by modifying natural tRNAs by incorporating 5-halouridines into the D-loop. In another example, PUS can modify uridines in the D-loop, t-psi-c loop, or anticodon loop (as well as uridines outside the D-loop, such as the tRNA stem). In other embodiments, inhibitors specific to PUS enzymes can be designed by modifying natural tRNAs by incorporating 5-halouridines into the D-loop, t-psi-c loop, and / or anticodon loop.

[0102] In some embodiments, the tRNA molecule comprises an anticodon arm and an acceptor arm, wherein the anticodon arm comprises a trinucleotide anticodon, and the anticodon recognizes a stop codon. In other embodiments, the anticodon recognizes a codon for alanine, arginine, aspartic acid, asparagine, cysteine, glycine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In one embodiment, the anticodon recognizes a cysteine ​​codon. In another embodiment, the acceptor arm comprises a 3'-terminal overhanging sequence of 5'-cytidine-cytidine-adenosine (CCA)-3'.

[0103] In some embodiments, the RNA molecule has the sequence: TIFF2026508359000003.tif10159, wherein one or more of any nucleotide, X, comprises an unnatural base, such as, for example, 5-halouracil (SEQ ID NO: 1). In other embodiments, the RNA molecule comprises the sequence of SEQ ID NO: 1, with the proviso that X is U, C, or G, and one or more U, C, or G of SEQ ID NO: 1 comprises an unnatural base.

[0104] In some embodiments, the RNA molecule comprises the sequence of a naturally occurring tRNA molecule, including, but not limited to, any one of the tRNA molecules listed in Table 2, except that the RNA molecule comprises at least one nucleotide at any one of the nucleotide positions that is capable of forming a covalent bond with a tRNA modifying enzyme. In some embodiments, at least one U, A, G, or C residue in the naturally occurring tRNA molecule is substituted with a 5-halo U, 8-halo G, 5-halo-C, 5-aza-C, or 8-aza-A. In some embodiments, the RNA molecule comprises or is identical to the parent tRNA with about 80% or more identity, e.g., about 85% or more identity, about 90% or more identity, about 92% or more identity, about 95% or more identity, about 97% or more identity, about 98% or more identity, or about 99% or more identity.

[0105] Table 2. Non-limiting examples of parent tRNA molecules for designing RNA-based inhibitors TIFF2026508359000004.tif59161TIFF2026508359000005.tif212161TIFF2026508359000006.tif222161TIFF2026508359000007.tif59161

[0106] In some embodiments, the RNA molecules of the invention comprise a certain percentage degree of sequence identity to any one of SEQ ID NOs: 1-53, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-53. In some embodiments, the specified degree of sequence identity retains one or more characteristics, e.g., a substrate for tRNA modifying enzymes and / or structure (e.g., cloverleaf secondary structure), as the RNA molecules of SEQ ID NOs: 1-53.

[0107] In some embodiments, the RNA molecule is an isolated or purified tRNA molecule.

[0108] In some embodiments, RNA molecules are purified.Contaminants can be the cellular proteins remaining after the RNA molecule of interest is expressed in a cell system, or the chemicals remaining after chemical synthesis.Suitable methods for purifying RNA molecules from contaminant mixtures are known in the art.In certain embodiments, the purity of the RNA molecules of the present invention is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure.

[0109] In one embodiment, an RNA molecule (eg, a tRNA molecule) comprises two or more unnatural bases, and the two or more unnatural bases inhibit two or more different tRNA-modifying enzymes.

[0110] In another aspect, the present invention provides compositions comprising the RNA molecules described herein.

[0111] In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. Suitable carriers and / or formulations for pharmaceutical compositions are described elsewhere herein.

[0112] method In one aspect, the present invention provides a method for inhibiting a tRNA modifying enzyme, the method comprising contacting a tRNA modifying enzyme in a cell with an RNA molecule described herein, wherein the formation of a covalent bond inhibits the tRNA modifying enzyme.

[0113] In some embodiments, the method is an in vitro method or an ex vivo method.

[0114] In one embodiment, at least one nucleotide comprises an unnatural base.

[0115] In another embodiment, the unnatural base comprises 5-halouracil or 8-azaadenosine.

[0116] In some embodiments, the non-naturally modified base is a 5-halouracil in the D-loop, the t-psi-c loop, and / or the anticodon loop of the tRNA molecule.

[0117] In other embodiments, the tRNA modifying enzyme is dihydrouridine synthase (DUS) or pseudouridine synthase (PUS).

[0118] In another aspect, the present invention provides a method for killing a cell, comprising contacting a tRNA-modifying enzyme in the cell with an RNA molecule described herein, wherein the formation of a covalent bond inhibits the tRNA-modifying enzyme, thereby killing the cell.

[0119] In some embodiments, the method is an in vitro method or an ex vivo method.

[0120] In one embodiment, the cell is a brain cancer cell, a gastrointestinal cancer cell, a kidney cancer cell, a liver cancer cell, or a lung cancer cell.

[0121] In another embodiment, the cells are cancer cells in culture.

[0122] In one embodiment, at least one nucleotide comprises an unnatural base.

[0123] In another embodiment, the unnatural base comprises 5-halouracil or 8-azaadenosine.

[0124] In some embodiments, the non-naturally modified base is a 5-halouracil in the D-loop, the t-psi-c loop, and / or the anticodon loop of the tRNA molecule.

[0125] In another aspect, the present invention provides a method for treating or preventing cancer in a subject in need thereof. The method comprises administering to the subject an effective amount of a pharmaceutical composition comprising an RNA molecule described herein. The RNA molecule contacts a tRNA-modifying enzyme in cancer cells of the cancer, and the formation of a covalent bond inhibits the tRNA-modifying enzyme, thereby killing the cancer cells.

[0126] In some aspects, the cancer is lung cancer, brain cancer, gastrointestinal cancer, kidney cancer, or liver cancer.

[0127] In other embodiments, the methods further comprise administering chemotherapy to the subject before, after, or simultaneously with administration of a pharmaceutical composition comprising an RNA molecule described herein.

[0128] In some embodiments, the subject is a mammal. In one embodiment, the subject is a human.

[0129] In another embodiment, the method comprises administering the pharmaceutical composition in combination with a therapeutic agent that induces ferroptosis in cancer cells.Ferroptosis-inducing compounds are described, for example, in Wang et al., Genes & Diseases, 9(2): 334-346 (March 2022), the entire contents of which are incorporated herein by reference.Non-limiting examples of ferroptosis-inducing compounds include, but are not limited to, GPX4 inhibitors, such as RSL3, ML162, ML210, and JKE-1674.

[0130] In some embodiments, the RNA molecules herein are further modified, conjugated, or combined with a delivery vehicle for administration to a subject in need thereof. Non-limiting modifications include, but are not limited to, backbone modifications, such as the introduction of phosphorothioate (PS) or phosphodiester (PO) linkages into the backbone of an RNA molecule; modification of nucleobases, for example, by the introduction of pyrimidine methylation (in addition to unnatural bases to inhibit tRNA-modifying enzymes), to improve delivery; modification of the ends of RNA molecules, for example, by the introduction of 5'-(E)-vinylphosphonate or abasic ribonucleotides to reduce exonuclease digestion, to improve delivery; modification of ribose sugar groups, for example, by the introduction of 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), and 2'-fluoro (2'-F) to improve the resistance of RNA molecules to nuclease digestion; conjugation of RNA molecules to lipids, peptides, aptamers, antibodies, or sugars to improve cellular uptake; or packaging of RNA molecules in nanoparticles. In some embodiments, the modification, conjugation, or delivery vehicle is the same as or similar to those already used to deliver oligonucleotide drugs, e.g., as described in Roberts et al. (Nature Reviews Drug Discovery volume 19, pp. 673-694 (2020)) and Huang et al. (Biomaterials Research volume 26, Article number: 49 (2022)).

[0131] Administration and Dosage The dosage regimen may affect what constitutes an effective amount. The therapeutic formulations contemplated in the present disclosure may be administered to a subject before or after the onset of a disease and / or disorder contemplated herein. Furthermore, several divided doses and staggered doses may be administered daily or continuously. Alternatively, the dose may be a continuous infusion or a bolus injection. Furthermore, the dosage of the therapeutic formulations contemplated in the present disclosure may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0132] The compositions contemplated within this disclosure can be administered to a patient, preferably a mammal, more preferably a human, using known procedures at a dosage and for a duration effective to treat the diseases and / or disorders contemplated herein in the patient. The effective amount of a therapeutic compound required to achieve a therapeutic effect may vary according to factors such as the state of the disease and / or disorder in the patient; the patient's age, sex, and weight; and the ability of a therapeutic compound contemplated within this disclosure to treat the diseases and / or disorders contemplated herein in the patient. Dosage regimens may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily. Alternatively, the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound contemplated within this disclosure is about 1 to 5,000 mg / kg body weight / day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of a therapeutic compound without undue experimentation.

[0133] Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated within this disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without toxicity to the patient.

[0134] In particular, the selected dosage level will depend on a variety of factors, including the activity of the particular compound used, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, or materials used in combination with the compound, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0135] A medical doctor, e.g., a physician or veterinarian, having ordinary skill in the art can readily determine the effective amount of pharmaceutical composition required and prescribe an effective amount of the pharmaceutical composition. For example, the physician or veterinarian could start dosing the compounds contemplated in the present disclosure used in the pharmaceutical composition at levels lower than those required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0136] In certain embodiments, it is particularly advantageous to formulate the compounds in unit dosage forms for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to a physically discrete unit suitable for unitary administration to the patient to be treated. Each unit contains a predetermined amount of therapeutic compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical vehicle. The unit dosage forms contemplated in the present disclosure are determined by and directly influenced by (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the constraints inherent in the field of compounding / formulating such therapeutic compounds to treat the diseases and / or disorders contemplated herein.

[0137] In certain embodiments, the compositions described herein are formulated with one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions described herein comprise a therapeutically effective amount of a compound described herein and a pharmaceutically acceptable carrier.

[0138] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. The activity of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of injectable compositions can be achieved by including absorption delaying agents, for example, aluminum monostearate and gelatin in the composition.

[0139] In certain embodiments, the compositions described herein are administered to a patient in dosages from one to five or more times daily. In other embodiments, the compositions described herein are administered to a patient in dosage ranges including, but not limited to, once daily, once every two days, once every three days, to once weekly, and once every two weeks. It will be readily apparent to those skilled in the art that the frequency of administration of the various combination compositions described herein will vary between individuals depending on many factors, including, but not limited to, age, the disease or disorder being treated, gender, general health, and other factors. Therefore, the present disclosure should not be construed as limited to any particular dosage regime, and the exact dosage and composition administered to any patient will be determined by the attending physician, taking all other factors into consideration.

[0140] The RNA molecule of the present invention for administration may be administered in amounts of about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg, about 200 μg to about 7,000 mg, about 3,050 μg to about 6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000 mg, about 1 mg to about 3,000 mg, about It may be within the range of 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, as well as any and all whole or partial increments therebetween.

[0141] In some embodiments, the administered dose of the RNA molecule is from about 1 mg to about 2,500 mg. In some embodiments, the dose used in the compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of the second compound described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0142] In certain aspects, the present disclosure relates to packaged pharmaceutical compositions comprising a container housing a therapeutically effective amount of an RNA molecule of the invention, alone or in combination with a second pharmaceutical agent, and instructions for treating or preventing, or reducing one or more symptoms of cancer in a subject.

[0143] The formulations may be used in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for intracranial, oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable administration method known in the art. Pharmaceutical preparations may be sterilized and, if desired, may be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring substances, flavoring substances, and / or aromatic substances. These may, if desired, be combined with other active agents, such as other analgesics.

[0144] Routes of administration of any of the compositions described herein include oral, nasal, rectal, vaginal, parenteral, buccal, sublingual, or topical administration. Compounds for use in the present disclosure may be formulated for administration by any suitable route, such as oral or parenteral administration, for example, transdermal, transmucosal (e.g., sublingual, lingual, (buccal), (urethral), vaginal (e.g., vaginal and perivaginal), nasal (intranasal), and rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

[0145] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, etc. It should be understood that the formulations and compositions useful in the present disclosure are not limited to the specific formulations and compositions described herein.

[0146] Oral administration For oral application, tablets, sugar-coated tablets, liquids, drops, suppositories, or capsules, caplets, and gel caps are particularly suitable.Compositions intended for oral use can be prepared according to any method known in the art, and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutical excipients suitable for tablet manufacture.Such excipients include, for example, inert diluents, such as lactose; granulating and disintegrating agents, such as corn starch; binders, such as starch; and lubricants, such as magnesium stearate.Tablets may be uncoated, or may be coated using known methods to ensure accurate or delayed release of active ingredients.Preparations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert diluent.

[0147] For oral administration, the compounds described herein may take the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients, such as binders (e.g., polyvinylpyrrolidone, hydroxypropyl cellulose, or hydroxypropylmethylcellulose); fillers (e.g., corn starch, lactose, microcrystalline cellulose, or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). If desired, tablets may be coated using appropriate methods and coating materials, such as the OPADRY™ film coating system available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY type, OYC type, Organic Enteric OY-P type, Aqueous Enteric OY-A type, OY-PM type, and OPADRY™ White, 32K18400). Liquid preparations for oral administration may take the form of solutions, syrups, or suspensions. Liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid).

[0148] The present disclosure also includes multi-layer tablets comprising a layer that provides delayed release of one or more compounds described herein and an additional layer that provides immediate release of another drug. A wax / pH-sensitive polymer mixture can be used to obtain a stomach-insoluble composition that entraps the active ingredient and ensures delayed release of the active ingredient.

[0149] Parenteral administration For parenteral administration, the RNA molecules described herein may be formulated for injection or infusion, e.g., intravenous, intramuscular, or subcutaneous, and may be formulated for administration in a bolus dose and / or continuous infusion. Suspensions, solutions, or emulsions in oily or aqueous vehicles, optionally containing other formulatory agents such as suspending, stabilizing, and / or dispersing agents, may also be used.

[0150] Further dosage forms Additional dosage forms of the present invention include those described in U.S. Patent Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of the present disclosure also include those described in U.S. Patent Application Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Further dosage forms of the present disclosure include those described in PCT Application Nos. WO03 / 35041; WO03 / 35040; WO03 / 35029; WO03 / 35177; WO03 / 35039; WO02 / 96404; WO02 / 32416; WO01 / 97783; WO01 / 56544; WO01 / 32217; WO98 / 55107; WO98 / 11879; WO97 / 47285; WO93 / 18755; and WO90 / 11757.

[0151] Sustained-Release Formulations and Drug Delivery Systems In certain embodiments, the formulations of the present invention may be, but are not limited to, short-acting formulations, rapid-offset formulations, and sustained-release formulations, including sustained-release formulations, delayed-release formulations, and pulsatile-release formulations.

[0152] The term sustained release is used in its conventional sense to refer to a drug formulation that gradually releases drug over an extended period of time, which may, but does not necessarily, result in substantially constant blood levels of drug over an extended period of time, which may be as long as a month or more and should result in a longer release than an equivalent amount of drug administered in bolus form.

[0153] For sustained release, the compounds may be formulated with suitable polymeric or hydrophobic materials that impart sustained release properties to the compounds. Thus, the compounds for use in the methods described herein may be administered in the form of microparticles, for example, by injection, or in the form of a wafer or disk by implantation.

[0154] In certain embodiments of the present disclosure, the RNA molecules described herein are administered to a patient using sustained release formulations, either alone or in combination with another pharmaceutical agent.

[0155] The term delayed release is used herein in its conventional sense to refer to a drug formulation that first releases the drug after some delay after the drug is administered, which may include, but is not necessarily, from about 10 minutes to about 12 hours.

[0156] The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that releases drug in a manner that produces a pulsatile plasma profile of the drug after the drug is administered.

[0157] The term immediate release is used in its conventional sense to refer to a drug formulation that releases the drug immediately after administration.

[0158] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any or all full or partial increments thereof, after the drug is administered.

[0159] As used herein, rapid off-action refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof, after the drug is administered.

[0160] dosage The therapeutically effective amount or dose of the RNA molecule of the present invention depends on the age, sex, weight, current medical condition of the patient, and the progression of cancer in the patient being treated. Those skilled in the art can determine the appropriate dosage depending on these and other factors.

[0161] Suitable doses of the RNA molecules described herein may range from about 0.01 mg to about 5,000 mg per day, e.g., from about 0.1 mg to about 1,000 mg, e.g., from about 1 mg to about 500 mg, e.g., from about 5 mg to about 250 mg per day. This dose may be administered in a single dose or in multiple doses, e.g., 1 to 4 or more times per day. When multiple doses are used, the amount of each dose may be the same or different. For example, a 1 mg dose per day may be administered as two 0.5 mg doses, with about 12 hours between doses.

[0162] It is understood that the amount of RNA molecule administered daily may, in non-limiting examples, be administered every day, every other day, every two days, every three days, every four days, or every five days. For example, when administered every other day, a 5 mg dose may be administered starting on Monday, a first 5 mg dose may be administered on Wednesday the following day, and a second 5 mg dose may be administered on Friday the following day.

[0163] If the subject's condition improves, optionally, the modulator of the present disclosure is administered continuously at the discretion of the physician. Alternatively, the dose of the administered drug is temporarily reduced or temporarily suspended for a certain period of time (i.e., a "drug holiday"). Optionally, the length of the drug holiday ranges from 2 days to 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reductions during drug holidays include, by way of example only, 10% to 100%, including 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

[0164] Once the patient's condition has improved, a maintenance dose is administered if necessary. Thereafter, the dosage and / or frequency of administration are reduced as a function of the patient's condition to a level at which the improved disease is maintained. In certain embodiments, if symptoms and / or infection recur, the patient will require intermittent treatment on a long-term basis.

[0165] The RNA molecules for use in the methods described herein can be formulated in unit dosage forms. The term "unit dosage form" refers to a physically discrete unit suitable as a unitary administration to a patient undergoing treatment. Each unit contains a predetermined amount of active material calculated to produce a desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or for one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0166] Optionally, LD 50 (the dose that causes death in 50% of the population) and ED 50The toxicity and therapeutic efficacy of such treatment regimens are determined in cell cultures or experimental animals, including, but not limited to, determination of the LD (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 and ED 50 Capsid assembly modulators that exhibit a large therapeutic index are preferred. Optionally, data obtained from cell culture assays and animal studies are used in formulating a dosage range for human use. The dosage of such capsid assembly modulators is preferably within the ED range with little or no toxicity. 50 Optionally, the dosage varies within this range depending upon the dosage form employed and the route of administration utilized. [Example]

[0167] The present invention will be further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless so specified. Therefore, the present invention should not be construed as being limited to the following examples, but rather should be construed to encompass any and all variations that become apparent as a result of the disclosure provided herein.

[0168] Without further explanation, one of ordinary skill in the art can, using the foregoing description and the following illustrative examples, make and utilize the molecules and compositions of the present invention and practice the claimed methods. Accordingly, the following working examples specifically point out aspects of the present invention and should not be construed as limiting.

[0169] Example 1: Design and testing of RNA-based inhibitors of tRNA-modifying enzymes To test the hypothesis that nonspecific chemotherapeutic compounds can be incorporated into tRNA molecules and inhibit tRNA-modifying enzymes by forming covalent bonds with them, we designed a nonspecific RNA molecule inhibitor based on natural tRNA molecules.

[0170] Referring to Figure 2, non-limiting RNA molecule inhibitors were designed that utilize the unnatural modified base 5-halouracil, which can react with the cysteine ​​residue in the active site of dihydrouridine synthase (DUS). Notably, the cysteine ​​residue in the active site is conserved in all DUSs (Rider et al., J Biol Chem. 2009 Apr 17;284(16):10324-33).

[0171] Referring to Figure 3, non-limiting examples of RNA molecule inhibitors herein are modified from natural tRNA (tRNACys) that decodes cysteine ​​codons. Uridine, which is located at position 20 (the 19th nucleoside) in tRNACys, is naturally modified to dihydrouridine by a tRNA-modifying enzyme such as DUS2. In the RNA molecule inhibitors, the uridine is replaced with 5-fluorouridine (5-FU), a non-natural modified nucleoside. The resulting RNA-based inhibitor is referred to as 5-FU tRNA or tRNACys-5FU. This non-limiting example of an RNA molecule inhibitor has the sequence shown in SEQ ID NO: 1.

[0172] Referring to FIG. 4, this study confirmed that RNA molecule inhibitors, including unnatural 5-FU nucleosides, can form stable covalent bonds with DUS2.

[0173] Referring to Figures 5 and 14A, the present study further demonstrated that tRNACys-5FU could significantly reduce the viability of lung cancer cell line PC9.

[0174] Example 2-1: Dihydrouridine synthase 2 maintains tRNACys ​​levels and prevents ferroptosis in lung cancer Dihydrouridine is a universally conserved tRNA modification incorporated by enzymes important for human health for reasons that remain unclear. High expression of dihydrouridine synthase 2 (DUS2) in lung adenocarcinoma predicts poor patient outcome. Here, this study shows that in human cells and mouse xenografts, DUS2 suppresses ferroptosis, a metal-dependent, non-apoptotic form of cell death. Many lung cancers are unusually sensitive to ferroptosis, and ferroptosis is emerging as a therapeutic target for lung cancer. Consistent with a positive role for DUS2 in lung adenocarcinoma growth and metastasis, high DUS2 expression correlates with increased resistance to ferroptosis inducers. Loss of DUS2 increases sensitivity with the concomitant accumulation of toxic lipid peroxides, a hallmark of ferroptotic cell death. Mechanistically, DUS2 is required to maintain levels of tRNA CysGCA and support the translation of cysteine-rich proteins, including metallothionein, which serve as key regulators of both metal and redox homeostasis. Metallothionein deficiency in DUS2 knockout cells leads to increased susceptibility to zinc toxicity and smaller reduced glutathione levels, which partially explains their susceptibility to ferroptosis. Our results reveal a tRNA-specific vulnerability and demonstrate the therapeutic potential of targeting DUS2.

[0175] Many cancers exhibit resistance to the classical apoptotic cell death pathway. Non-small cell lung cancer (NSCLC) uses multiple mechanisms to evade apoptosis, including loss of expression of the pro-apoptotic gene Bcl-2-like protein (BIM) and amplification of the anti-apoptotic gene induced myeloid leukemia cell differentiation protein (MCL1). One such cell death mechanism, ferroptosis, is a form of non-apoptotic cell death that is emerging as a therapeutic target for lung cancer.

[0176] Example 2-2: DUS2 is overexpressed in lung cancer, and loss of DUS2 sensitizes cells to ferroptosis Hallmarks of ferroptotic cell death include a dependence on redox-active iron and the accumulation of toxic lipid peroxides. Several compounds have been described to induce ferroptosis, including class I ferroptosis inducers that inhibit cystine (erastin) import and class II ferroptosis inducers ((1S,3R)-RSL3, M162, and ML210) that inhibit the activity of the phospholipid hydroperoxidase GPX4 (Figure 6A). Several studies have demonstrated that NSCLC cell lines are sensitive to chemical ferroptosis inducers both in vitro and in vivo, and the development of ferroptosis-modulating drugs is an active area of ​​research. Interestingly, in a panel of 860 cancer cell lines, resistance to treatment with several class II ferroptosis inducers (RSL3, ML162, and ML210) correlated with expression of the tRNA-modifying enzyme dihydrouridine synthase 2 (DUS2) (Figure 6B).

[0177] Dihydrouridine synthase (DUS) incorporates modified uridine forms into RNA (Figure 6C). Dihydrouridine (D) is the most common modified nucleotide in tRNA, found in tRNAs from organisms from all branches of the tree of life. D is thought to stabilize the correct folding of the D-loop in tRNA. Eukaryotes, including humans, express four D synthases, each with a unique target nucleotide in multiple individual tRNAs. Disruptions in D levels and / or DUS expression have been linked to lung, brain, and kidney cancers. DUS2 is known to modify tRNAs at a position within the tRNA D-loop in yeast. DUS2 is frequently overexpressed in non-small cell lung cancer (NSCLC) tumors (Figure 6D), and survival times of NSCLC patients whose tumors express high levels of DUS2 are shorter than those whose tumors do not express high levels of DUS2 (Figure 6E).

[0178] Here, we investigated the role of DUS2 in NSCLC disease progression. We used CRISPR / Cas9 to knock out DUS2 in an NSCLC cell line (A549) that expresses high levels of DUS2. Loss of DUS2 confers hypersensitivity to ferroptosis-inducing compounds. Consistent with the in vitro sensitivity of DUS2 KO to ferroptosis, this study shows that DUS2 KO cells form smaller tumors in a mouse xenograft model and are more sensitive to systemic administration of ferroptosis inducers. We also explored the role of DUS2 in gene expression. Loss of DUS2 results in a 40% reduction in the levels of one tRNA, CysGCA. This loss of tRNACysGCA leads to a proteome-wide reduction in cysteine-rich protein levels, including a reduction in the synthesis of a family of small, highly conserved cysteine-rich proteins called metallothioneins (MTs). MT is known to inhibit ferroptosis and plays two important roles in cells. First, MT directly inhibits the formation of lipid peroxides, protecting cells from oxidative stress. Second, MT is a major regulator of intracellular zinc levels. Our results establish a promising basis for increased ferroptosis in the absence of DUS2 upon loss of MT expression and suggest the therapeutic potential of targeting DUS2 in lung cancer.

[0179] To investigate the association between high DUS2 expression in NSCLC and poor patient prognosis, this study used CRISPR / Cas9 to generate knockout (KO) cell lines in a common NSCLC model cell line (A549) that expresses high levels of DUS2. Multiple independent clonal KO cell lines were recovered using two different lentiviral-delivered guide RNAs targeting exons 3 and 4 of the DUS2 coding sequence. As expected, the clonal DUS2 KO CRISPR lines had no detectable DUS2 protein expression (Figure 6F).

[0180] DUS2 mRNA levels correlated with resistance to known ferroptosis-inducing compounds across a panel of 860 cell lines, prompting us to test the susceptibility of DUS2 KO cells to ferroptosis. DUS2 KO cells showed a roughly two-fold increase in the percentage of dead cells after ferroptosis induction with the GPX4 inhibitor RSL3 (Figure 6G). This sensitivity was reversed upon DUS2 re-expression (Figure 10A) or treatment with the known ferroptosis inhibitors Trolox and Ferrostatin (Figures 10A-10C), but not with the apoptosis inhibitor Z-FAD-FMK (Figures 10A-10C). A key feature of ferroptotic cell death is the accumulation of toxic lipid peroxides. In this study, we measured lipid peroxidation levels after GPX4 inhibitor treatment using the oxidation-sensitive fluorescent lipid peroxidation probe C11-BODIPY26. Compared to WT A549 cells, DUS2 KO cells exhibited 6- to 8-fold higher lipid peroxidation levels upon treatment with RSL3 (Figures 6H-6I) or a second GPX4 inhibitor, ML162 (Figure 10D). Consistent with the higher levels of lipid peroxidation in DUS2 KO cells, we observed elevated cellular reactive oxygen species (ROS) levels upon treatment of DUS2 KO cells with RSL3 using the probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (Figure 10E).

[0181] Example 2-3: DUS2 is required to maintain the levels of a specific tRNA, tRNACysGCA DUS2 is known to modify tRNA at position 20 in the tRNA D-loop in yeast (Figure 7A), where D is known to stabilize tRNA folding. Therefore, in this study, we next investigated whether DUS2 is required for maintaining tRNA expression or function in NSCLC cells. We performed tRNA sequencing using a combination of ARM-seq and DM-TIGRT-seq protocols. DUS2 KO cells showed no significant changes in the charge fraction of any tRNA. As a positive control, a 70% reduction in tRNA Gln charge was detected after glutamine depletion (Figure 7B). Notably, both DUS2 KO cell lines showed a reproducible reduction in the expression of nearly all tRNA CysGCA isoencoders expressed in A549 cells (Figures 7C-7D). When tRNACysGCA levels are summed across all isocoders, DUS2 KO cells show a reduction of approximately 40% in the total pool of tRNACysGCA (Fig. 11).

[0182] Next, we reanalyzed small RNA-seq data from TCGA lung adenocarcinoma (LUAD) samples for tRNA expression. We found a statistically significant increase in the levels of tRNACysGCA in patient tumor samples compared with non-tumor samples (Figure 7E). The levels of another U20-containing tRNA (tRNAGlnCTG) in tumor samples were not significantly different (Figure 7F). Collectively, these results demonstrate that DUS2 is required to maintain the levels of a specific tRNA, tRNACysGCA, in NSCLC cells, suggesting a role for tRNACysGCA levels in lung cancer disease.

[0183] Example 2-4: Loss of DUS2 impairs the translation of cysteine-rich proteins, including known anti-ferroptosis oncoproteins To determine whether the approximately 40% reduction in tRNACysGCA expression in DUS2 KO cells functionally impacts translation, we measured cysteine ​​codon translation using a luciferase reporter. A series of 15 cysteine ​​codons was added before the P2A sequence, followed by firefly luciferase as a surrogate for cysteine ​​repeat peptide production and IRES-driven Renilla luciferase as a normalization control (Figure 8A). Because cysteine ​​is encoded by two independent codons (UGU and UGC) encoded by the same GCA anticodon tRNA pool, we generated versions of the cysteine ​​repeat reporter with either the UGU or UGC codon. When introduced into DUS2 KO cells by transfection, these reporters demonstrated a 40% reduction in the ratio of firefly luciferase to Renilla luciferase (Figure 8B). This loss of efficient cysteine ​​translation was partially rescued by transfection of DUS2 KO cells with in vitro transcribed tRNACysGCA (Fig. 8C). The observed defect in cysteine ​​translation did not affect bulk protein synthesis, as confirmed by 35S methionine incorporation (Fig. S12A).

[0184] Next, we used SILAC proteomics to measure changes in endogenous protein levels in DUS2 KO cells. Consistent with a defect in cysteine ​​codon translation, the abundance of proteins with a Cys content greater than 5% was significantly reduced in cells lacking DUS2 when analyzed by amino acid content (Figure 8D). Due to the inherently limited coverage of shotgun proteomics and the fact that many cysteine-rich proteins are secreted, SILAC experiments detected only relatively abundant proteins with moderate cysteine ​​content. In this study, we did not observe peptides corresponding to many cysteine-rich proteins, including any metallothioneins. Metallothioneins (MTs) are a class of highly cysteine-rich (approximately 35% cysteine ​​content) proteins that have been linked to ferroptosis and cancer progression. Therefore, in this study, we measured metallothionein translation in DUS2 KO cells using a dual-luciferase reporter similar to a cysteine ​​codon repeat reporter by exchanging a series of cysteine ​​codons with the coding sequence of MT1A or MT1G (Figure 8E). Metallothionein protein production was impaired in DUS2 KO cells (Figures 8G-8H). This demonstrates that loss of DUS2 activity leads to defective production of endogenously expressed cysteine-rich oncoproteins known to inhibit ferroptosis and lipid peroxidation.

[0185] In parallel, we measured steady-state mRNA levels in DUS2 KO cells by RNA-seq. Among the hundreds of mRNAs differentially expressed in DUS2 KO cells (Figure 12B), we noticed a decrease in mRNAs encoding cysteine-rich proteins in DUS2 KO cells (Figure 12B). It has been shown that slow translation elongation induces mRNA degradation downstream of surveillance by the ribosome quality control (RQC) pathway. We hypothesized that reduced tRNACysGCA levels in DUS2 KO cells impair cysteine ​​translation, leading to more frequent ribosome stalling at cysteine ​​codons and resulting in RQC-mediated cysteine-rich mRNA decay. Supporting this hypothesis, metallothionein mRNA abundance was reduced in DUS2 KO cells and was partially restored by knocking down the RQC factor GIGYF2 (Figure 12C).

[0186] Metallothionein utilizes its high thiol content to fulfill two interrelated roles in cells. First, metallothionein binds Zn 2+ and Cu 2+ DUS2 is a key regulator of cellular zinc and copper levels by directly coordinating to ions. Ferroptosis was first characterized as an iron-dependent form of cell death, but more recent studies have shown that defects in zinc homeostasis, which elevate cytosolic zinc concentrations, sensitize cells to ferroptosis. Given the established role of metallothionein in regulating intracellular zinc levels, the role of zinc in the ferroptosis susceptibility of cells lacking DUS2 was explored. DUS2 KO cells exhibited a Zn 2+ Poisoning and Zn 2+DUS2 KO cells are more susceptible to ferroptosis-induced cell death (Figure 8G). A second function of MTs is to protect cells from oxidative stress and directly inhibit the accumulation of lipid peroxides, a hallmark of ferroptosis. We hypothesized that the reduced MT levels in DUS2 KO cells increase the demand for lipid peroxide reduction via GPX4, resulting in less cellular glutathione (GSH) reduction. Supporting this hypothesis, we found that DUS2 KO cells have significantly lower GSH levels (Figure 8H). Together, these observations suggest that the primary cause of ferroptosis susceptibility in DUS2 KO cells is the loss of MT synthesis, resulting in defects in metal and redox homeostasis (Figure 8I).

[0187] Example 2-5: Combining DUS2 loss with ferroptosis induction extends lifespan in a mouse xenograft NSCLC model To characterize the effects of DUS2 on tumor growth and progression in vivo, we subcutaneously injected A549 or DUS2 KO cells to develop xenograft tumors in nude mice. After injection, we monitored tumor size and mouse survival. Tumors derived from DUS2 KO cells took 33% longer to establish and were significantly smaller than tumors derived from A549 cells (Figures 9A-9B). Examination of DUS2 WT and KO tumors revealed that DUS2 KO tumors showed a moderate increase in the expression of the ferroptosis biomarker PTSG2, suggesting that ferroptosis is endogenously induced in tumors (Figure 9C). Ferroptosis inducers are a promising therapeutic approach for treating some cancers. In vitro experiments suggest that ferroptosis induction, when combined with DUS2 inhibition, may be a more effective NSCLC treatment strategy. To determine whether the ferroptosis sensitivity of DUS2 KO cells could be exploited for therapeutic benefit, we induced ferroptosis in mice bearing established tumors by administering a GPX4 inhibitor (Figure 9D). Most GPX4 inhibitors (RSL3, ML162, and ML210) suffer from poor pharmacological properties, limiting their in vivo utility. However, a new class of GPX4 inhibitors with improved physiochemical and pharmacokinetic properties has recently been developed. In this study, we first tested whether oral administration of one of these compounds (JKE-1674) could induce ferroptosis in mouse lungs by measuring the mRNA levels of the ferroptosis marker PTSG241 after oral JKE-1674 administration. JKE-1674 induced PTSG2 mRNA in lung tissue by approximately 8.5-fold (Figure 9E), which was similar to the level induced by other GPX4 inhibitors. JKE-1674 treatment induced PTSG2 expression threefold in DUS2 KO tumors (Figure ​(Figure9F). 9F). Among JKE-treated mice, mice bearing DUS2 KO tumors had a significantly longer lifespan (Figure ​(Figure9G). 9G). Taken together, these data suggest that either DUS2 inhibition or a combination of DUS2 inhibition and ferroptosis induction may be a promising therapeutic strategy for treating NSCLC patients.

[0188] Examples 2-6 Our data demonstrate that high expression of ubiquitous tRNA-modifying enzymes represents a specific cancer vulnerability in NSCLC cells. DUS2 is frequently overexpressed in NSCLC, and patients whose tumors express high levels of DUS2 have poorer outcomes. Using DUS2-depleted NSCLC cells, we demonstrate that DUS2 is required to support the levels of a specific tRNA family, tRNA CysGCA. This result highlights the outsized role that specific tRNA substrates may play in the biological function of tRNA-modifying enzymes. Loss of CysGCA expression in DUS2 KO cells leads to defects in the translation of cysteine ​​codons, thereby reducing the steady-state levels of many cysteine-rich proteins, including metallothionein, which plays an important role in regulating cellular zinc levels and responding to oxidative stress. Loss of metallothionein expression in DUS2 KO cells sensitizes the cells to ferroptosis both in vitro and in vivo.

[0189] Our data support the need for cysteine ​​incorporation into both GSH and cysteine-rich metallothionein proteins in lung cancer cells to avoid ferroptosis. Inhibition of either DUS2 or the MT family increased ferroptosis susceptibility in patients and retained therapeutic value.

[0190] Examples 2-7: Methods cell culture A549 cells were maintained in a 50:50 mixture of DMEM:F12 medium (Gibco) supplemented with 1x penicillin / streptomycin (Gibco) and 10% FBS (Sigma). Cells were grown at 37°C in 5% CO and maintained at subconfluency.

[0191] CRISPR knockout generation To delete the third and fourth exons of DUS2, we generated DUS2 CRISPR knockout A549 cells using the single-guide LentiCRISPRv2 strategy. Oligonucleotides for each guide RNA were phosphorylated, annealed, and then cloned into BsmBI-digested pLentiGuide-Puro (Addgene). Cas9 / guide RNA lentivirus was generated by transfecting 293T cells with pLentiGuide-Puro, psPAX2 (Addgene), and pdr8.2 (Addgene). Viral supernatants were collected, filtered, and flash-frozen 48 and 96 hours after transfection. For infection, 1 mL of the 48-hour viral supernatant was placed in a 6-well dish with A549 cells at 50% confluency. At 90% confluency, A549 cells were split into 10 cm dishes and selected for stable integration using 1 μg / mL puromycin (Sigma). After generating stable puro-resistant populations, single clones were isolated using serial dilution and colony picking. Single cell clones were expanded, screened for lack of DUS2 protein expression, and frozen.

[0192] Western blotting A549 cells were pelleted and whole-cell lysates were prepared by resuspending fresh or frozen (-80°C) pellets in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% NP-40) and vortexing on ice for 10 minutes. Lysates were clarified by centrifugation at 4°C and maximum speed (22,500 x g) for 15 minutes. Approximately 20 μg of whole-cell lysate, as determined by BCA assay, was run on a 7% Tris-acetate gel and transferred to a nitrocellulose membrane using wet transfer. The membrane was blocked with 5% milk for 1 hour and incubated overnight at 4°C with primary antibody dissolved in 5% milk low-salt TBST (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween-20). The antibodies used for Western blotting were as follows: anti-DUS2 at 1:10,000 and anti-GAPDH (Sigma-Aldrich G9545) at 1:10,000. Secondary antibody incubation was performed with HRP-conjugated goat anti-rabbit IgG (Promega W4011) at 1:3000 for 1 hour at room temperature. Washes were performed with high-salt TBST (50 mM Tris pH 7.5, 400 mM NaCl, 0.1% Tween-20).

[0193] AlkB and AlkB D135S purification pET30a-AlkB and pET30a-AlkB(D135S) (Addgene) were transformed into BL21(DE3) (NEB). 1 L cultures were grown at 37°C with shaking to an OD of 0.55. IPTG (Gold Bio) and FeSO4 (Sigma) were added to 1 mM and 10 μM final concentrations. Cultures were induced at 37°C with shaking for 4 hours, and cells were harvested by centrifugation and flash-frozen. Each 1 L pellet was resuspended in 20 mL of fresh AlkB lysis buffer (50 mM HEPES pH 8.0, 10 mM Fe(II) sulfate, 300 mM NaCl, and 5 mM imidazole). Cells were lysed by sonication and the addition of lysozyme (Sigma). Lysates were clarified by spinning at 12,000 x g for 30 minutes at 4°C. The lysate was filtered through a 0.2 μM filter and loaded onto a HisTrap 5 mL nickel column (Cytiva). Unbound protein and RNA were removed by extensive washing with lysis buffer, and crude AlkB protein was eluted from the Ni column using AlkB lysis buffer containing 250 mM imidazole. Fractions containing AlkB protein were pooled and desalted using a Zeba spin desalting column (Thermo). The desalted protein was purified from the bound RNA using a MonoS column (Cytiva) with a 100 mM to 1 M NaCl gradient. Fractions containing AlkB protein were pooled and concentrated using an Amicon Ultra-15 10K MWCO filter (Milipore). The concentrated AlkB protein was fractionated on a HiLoad 16 / 60 Superdex S200 column (Cytiva). The S200 fraction containing AlkB was reconcentrated using an Amicon Ultra-15 10K MWCO filter (Milipore), diluted to 50% glycerol, and flash-frozen.

[0194] Total RNA isolation A549 cells were harvested by pelleting and resuspending fresh or frozen (-80°C) pellets in 1 mL of QIAzol (Qiagen). Total RNA was harvested according to the manufacturer's protocol.

[0195] tRNA sequencing Total RNA from A549 cells was resuspended in 100 mM NaOAc / HOAc pH 4.8. 3 μL of 1 M NaIO4 (50 mM FC) was added, and the mixture was incubated at 22°C. After 30 minutes, 6.65 μL of 1 M glucose was added. Total RNA was then recovered by EtOH precipitation. Briefly, 10 μL of 3 M NaOAc, 1 mL EtOH was added, and the mixture was incubated at -20°C for 15 minutes, followed by spinning at 4°C and maximum speed (22,500 x g) for 30 minutes. The RNA pellet was washed with 70% EtOH and respun for 5 minutes. The pellet was resuspended in 50 μL of Na borate pH 9.5 and incubated at 45°C for 90 minutes. Large RNAs were depleted from the total RNA using a Qiagen miRNeasy spin column according to the manufacturer's recommendations. Small RNAs were demethylated with AlkB and AlkB D135S in AlkB buffer (50 mM HEPES KOH, pH 8, 75 μM ammonium ferric sulfate pH 5, 1 mM α-ketoglutarate, 2 mM sodium ascorbate, 50 μg / ml BSA) at 37°C for 100 min with a 4x molar ratio of wtAlkB and 4x molar ratio of D135S. The RNA was recovered by modified Silane bead purification. The RNA was then 3'-end healed using T4 PNK (NEB) and CIP (NEB) and re-recovered by modified Silane bead purification. 3' adapters were ligated to the small RNAs using T4Rnl2. 5 μL of RNA was incubated with 1.5 μL of DMSO and 0.5 μL of 80 μM preadenylated 3' adapter. The mixture was incubated at 65°C for 2 min and placed on ice for 1 min. The ligation was incubated overnight at 16°C with 3.5 μL of water, 2 μL of 10X NEB Ligase 50 buffer, 5 μL of 50% PEG 8000, 1 μL of Superasin (Thermo), and 2 μL of RNA ligase (NEB). RNA was recovered by denaturing silane bead purification. RNA was reverse transcribed using Superscript III. 8 μL of RNA was annealed with the RT primer at 65°C for 5 minutes and cooled to RT on the benchtop for 10 minutes. RT was performed according to the manufacturer's instructions.The RT reaction was added with 1 μL of 1M NaOH and incubated at 95°C for 5 minutes. RNA was removed from the cDNA by adding 1 μL of 1M HCl. The cDNA was recovered by modified Silane bead purification. A 5' linker was ligated to the cDNA using T4 RNA ligase. The cDNA was mixed with 0.8 μL of 80 μM 5' adapter and 1 μL of DMSO. The mixture was incubated at 75°C for 2 minutes and placed on ice for 1 minute. To this, 4.6 μL of water, 2 μL of 10X NEB RNA ligase buffer, 0.2 μL of 0.1M ATP, 5 μL of 50% PEG 8000, and 2 μL of RNA ligase were added. The mixture was incubated overnight at RT with shaking. The linker-ligated cDNA was recovered by modified Silane bead purification. Final library PCR was performed using Phusion DNA polymerase according to the manufacturer's recommendations.

[0196] tRNA rescue experiments T7 template DNA was constructed using PCR to fuse the T7 promoter sequence with the tRNA CysGCA sequence by adding a CCA tail. tRNA CysGCA RNA was prepared by run-off transcription using T7 RNAP at 37°C for 8 hours, followed by template removal using DNAse I (Ambion) at 37°C for 30 minutes. Full-length tRNA CysGCA was purified on an 8% denaturing urea-PAGE gel, eluted overnight, ethanol precipitated, and resuspended in water. For rescue experiments, 2 μg of tRNA CysGCA and 2 μg of a translation reporter plasmid were cotransfected into cells using TransIT-X2 (Mirus). Forty-eight (48) hours after transfection, cells were lysed in 500 μL of 1X passive lysis buffer (Promega), harvested, and flash-frozen. The lysates were frozen and thawed twice, and 75 μL of the lysates were used to measure firefly luciferase activity and Renilla luciferase activity using a dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions.

[0197] DUS2 rescue experiment Full-length DUS2 was cloned into pcDNA3.1 (CMV promoter, C-terminal FLAG tag), and 2 µg of DUS2 plasmid was transfected into cells using TransIT-X2 (Mirus). Forty-eight (48) hours after transfection, cells were split into 6-well plates and allowed to recover for 24 hours. At 40-50% confluency, cells were treated with the indicated concentrations of ferroptosis-inducing compounds for 12 hours. Cells were then stained with annexin / PI as follows:

[0198] tRNAseq data analysis Demultiplexed reads were adapter trimmed using BBTools bbduk.sh. The adapter-trimmed reads were then PCR-duplicate collapsed based on unique molecular identifiers (UMIs) using dedupe.sh. The UMIs were then force trimmed by a second trimming. The adapter-trimmed and duplicate-collapsed reads were then aligned to one copy of each isodecoder pseudogenome using bbmap.sh. tRNA expression was quantified by counting the number of uniquely mapped reads that mapped to tRNAs, and differential expression analysis was performed using limma-voom. tRNAs with fewer than 100 uniquely mapped reads were not considered during expression analysis. A custom Python script was used to calculate the tRNA charge ratio, expressed as the ratio of the number of reads terminating in CC-3' or CCA-3'.

[0199] 35 S Met total protein synthesis Equal amounts of DUS2 KO and wt cells were seeded in 6-well plates. Cells were grown to approximately 80% confluency, and the medium was switched to DMEM-Met for 20 minutes. 10 μL of 100 μCi / mL 35S Met was added to each well and incubated at 37°C for 30 minutes. To harvest, cells were washed twice with 1X PBS and lysed in 200 μL of RIPA containing 1X PMSF and 1X Complete. Lysates were freeze-thawed twice and spun at 22,500 x g for 15 minutes at 4°C to pellet cellular debris. Equal amounts of whole-cell lysates, as confirmed by BCA assay, were loaded onto a 4-20% SDS-PAGE gel, dried for 2 hours, and exposed overnight to a storage phosphor screen.

[0200] Dual luciferase assay The pCMV:codonarray:P2A:Fluc:IRES:Rluc or pCMV:metallothionein:P2A:Fluc:IRES:Rluc constructs were assembled by Gibson assembly into pTwist CMV Hygro, and successful assembly was confirmed by Sanger sequencing. Two micrograms of each plasmid was transfected into cells using TransIT-X2 (Mirus). Forty-eight (48) hours after transfection, cells were lysed in 1X passive lysis buffer (Promega), harvested, and flash-frozen. Lysates were freeze-thawed twice, and then 75 μL of lysate was used to measure firefly luciferase and Renilla luciferase activities using a dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions.

[0201] SILAC proteomics SILAC experiments were configured as two-channel experiments. Cells were grown in 1:1 DMEM:F12 containing dialyzed FBS (Gibco) supplemented with either unlabeled Arg and Lys (Invitrogen) or 13C6,15N4 Arg and 13C6,15N2 Lys (Invitrogen). Cells were maintained in isotope-labeled medium for 10 doublings and then harvested by lysis in RIPA supplemented with 1 mM PMSF and 1X HALT phosphatase / protease cocktail (Pierce). Lysates were clarified at 4°C and 22,500 x g for 10 min. Total protein was quantified using the BCA assay, and 120 μg of total protein was submitted to the Yale MS & Proteomics Resource for processing and analysis. Total protein samples were filtered through a 3-kDa Amicon Ultra filter, and the retentate was dried in a SpeedVac and used for downstream proteomics preparation. The dried protein pellet was reduced with DTT, alkylated with iodoacetamide, enzymatically digested with trypsin, and desalted using a C18 RP microspin column. High-resolution liquid chromatography-mass spectrometry (MS / MS) data were collected on an Orbitrap Fusion mass spectrometer coupled to a NanoACQUITY UPLC. All MS / MS samples were analyzed using Mascot (Matrix Science, Mascot version 2.7.0). For peptide identification, Mascot was configured to search SwissProt assuming the digestion enzyme trypsin. Mascot searches were performed using a fragment ion mass tolerance of 0.020 Da and a parent ion tolerance of 10.0 PPM. MS / MS-based peptide and protein identifications were validated using Scaffold (version 4.11.1, Proteome Software). Peptide identifications were accepted if they were confirmed with a probability of >95.0% by the Scaffold Local FDR algorithm.Protein identifications were accepted if they could be confirmed with a probability of >99.0% and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.

[0202] RNAseq Total RNA was isolated from three replicates of A549 and both DUS2 KO cell lines as described above. Stranded poly(A)+-selected mRNA-seq libraries were prepared by Genewiz and sequenced on a HiSeq X 10 using paired-end 150-bp reads.

[0203] qRT-PCR Total RNA was isolated as described previously. For siRNA knockdown experiments, cells were seeded into 6-well plates and transfected with siGIGFY2 or siNT siRNA for 48 hours using TransIT-X2 (Mirus). Total RNA was DNase-treated using TURBO DNAse (Thermo) according to the manufacturer's instructions. One-step qRT-PCR was performed using gene-specific forward and reverse primers and Luna Universal One-Step RT-qPCR (NEB) reagents in a CFX96 Real-Time PCR instrument (Bio-Rad). Fold changes were calculated using the Pfaffl method with GAPDH as the housekeeping gene. For qPCR experiments from tissues and tumors, cells were dissociated, pelleted, and resuspended in TRIzol (Invitrogen). RNA was then extracted according to the manufacturer's instructions.

[0204] Cell death measurement by Annexin V / propidium iodide staining Cells were counted and seeded into 6-well plates (Corning). At 40-50% confluency, cells were treated with ferroptosis-inducing or ferroptosis-inhibiting compounds (RSL3, Cayman Chemical; ML162, Cayman Chemical; Trolox, Sigma; ZVAD-FMK, Promega; ZnCl2, Sigma) for 12 hours. Cells were harvested by trypsinization and centrifugation, washed once with 1X Hank's buffered salt solution (HBSS), resuspended in 1X annexin-binding buffer (Thermo), and stained with annexin V / propidium iodide according to the manufacturer's instructions. Cells were filtered through a 70-micron filter and analyzed on a BD LSR II FACS analyzer using FITC and propidium iodide filter sets.

[0205] Quantification of lipid oxidation using C11-BODIPY staining Cells were counted and seeded into 6-well plates (Corning). At 40-50% confluency, cells were treated with the indicated concentrations of ferroptosis-inducing compounds for 12 hours, followed by treatment with 1 μM C11-BODIPY for 30 minutes. Cells were then harvested by trypsinization and centrifugation, washed once with 1X HBSS, and resuspended in 1X Dulbecco's phosphate-buffered saline (DPBS). Cells were filtered through a 70-micron filter and analyzed on a BD LSR II FACS analyzer using FITC (reduced C11-BODIPY) or PE (oxidized C11-BODIPY) filter sets.

[0206] Measurement of cellular glutathione concentration Cells were seeded into black 96-well cell culture-treated plates (Corning). At 80% confluency, the medium was removed and the cells were washed once with 1X DPBS. Glutathione levels were measured using GSH-Glo reagent (Promega) according to the manufacturer's instructions.

[0207] Subcutaneous mouse xenografts All animal protocols were reviewed by the Yale University IACUC and approved under protocol 2020-20303. A549 cells and A549 DUS2 KO cells were washed with 1X PBS, resuspended in 1X PBS, and combined 1:1 with Matrigel (Corning) to a concentration of 5,000,000 cells / mL. Mice were randomized prior to injection. 500,000 A549 cells (100 μL) were injected subcutaneously into both flanks of six female nude mice. 500,000 A549 DUS2 KO cells (100 μL) were injected subcutaneously into both flanks of six female nude mice. Mice were anesthetized with isoflurane twice weekly, during which time mice were weighed and tumor volumes were measured. Tumors were measured in two dimensions with calipers, and tumor volume was calculated using the following formula: Volume = 0.5 × L1 × L2. 2 , where L1>L2.

[0208] Dosing of each mouse began when at least one dimension of the tumor reached at least 5 mm in length. Mice were randomized within each group prior to dosing. Mice were given 10 mg / mL JKE-1674 (MedChemExpress) (10% 100 mg / mL JKE-1674 in DMSO, 90% 20% β-cyclodextran in 1X PBS) by oral gavage to a concentration of 50 mg JKE-1674 per kg body weight, or vehicle solution (10% DMSO, 90% 20% β-cyclodextran in 1X PBS). Three of the six mice injected with A549 cells or A549 DUS2 KO cells alone were dosed with JKE-1674, while the remaining mice were dosed with vehicle solution. Mice were dosed twice weekly. Survival endpoints are defined by death (naturally or as determined by a veterinary technician based on the health of each mouse), 15% weight loss, or tumors reaching 2 cm in length in any dimension.

[0209] Example 3: The studies described in Example 3 investigated the effects of non-limiting examples of RNA-based DUS / PUS inhibitors herein on various cell types.

[0210] In Example 3, HepG2 cells were maintained in DMEM + 10% FBS. A549 cells were maintained in 50:50 DMEM:F12 + 10% FBS. CLB-001 tRNA was transfected using Lipofectamine RNAiMAX. Cell viability was measured using Cell TiterGlo 2.0.

[0211] Example 3-1: RNA-based inhibitors of tRNA-modifying enzymes kill cancer cells but not non-transformed cells Referring to Figures 14A and 14B, a non-limiting example of an RNA-based inhibitor of the DUS enzyme described in Example 1 (tRNACys ​​having the sequence shown in SEQ ID NO: 1, in which all uridine residues have been replaced with the unnatural modified nucleoside 5-flurouradine (5-FU), and referred to as CLB-001 in the Example 3 section) exhibited potent IC 50 It was found that it can kill two different types of cancer cells.

[0212] Referring to Figure 14A, cultured hepatocellular carcinoma cell line HepG2 was treated with various concentrations of CLB-001. It was found that high concentrations of CLB-001 could almost completely kill hepatocellular carcinoma cells. The IC50 was calculated to be approximately 5 nM.

[0213] Referring to Figure 14B, cultured non-small cell lung cancer cell line A549 was treated with various concentrations of CLB-001. It was found that high concentrations of CLB-001 could almost completely kill non-small cell lung cancer cells. The IC50 was calculated to be approximately 10 nM.

[0214] Referring to Figure 19, cultured hepatocellular carcinoma cell lines PLC / PRF / 5 and SNU-387 were treated with various concentrations of CLB-001. High concentrations of CLB-001 were found to be able to almost completely kill hepatocellular carcinoma cells. The IC50 was calculated to be between 3 nM and 30 nM.

[0215] Notably, CLB-001 did not show significant toxicity to non-cancer cells. Referring to Figure 15, when the non-transformed hepatocyte cell line AML12 was subjected to various concentrations of CLB-001, no statistically significant cell death caused by the DUS inhibitor was observed. This was true even at a relatively high concentration of 1000 nM CLB-001.

[0216] Example 3-2: CLB-001 was much more potent than 5-fluorouracil in killing cancer cells This study found that CLB-001 was much more potent than 5FU in killing HepG2 cancer cells.

[0217] Referring to Figure 16, HepG2 cells were divided into two groups. The first group was subjected to various concentrations of 5-fluorouracil (5FU), and the second group was subjected to various concentrations of CLB-001, in which 5FU was incorporated into tRNACys ​​molecules. The experiment demonstrates that CLB-001 is over 7000 times more potent than 5FU in killing HepG2 cancer cells.

[0218] Example 3-3: High tRNA-modifying enzyme levels are associated with hepatocellular carcinoma and poor outcome in hepatocellular carcinoma Referring to Figures 17A-17C, various modifying enzymes (PUS1, PUSL1, PUS7, RPUSD1, RPUSD2, TRMT2A, TRMT2B, DUS1L, DUS2, DUS3L, and DUS4L) are upregulated in hepatocellular carcinoma tumors as assayed by the mRNA levels of these enzymes.

[0219] Referring to Figures 18A-18G, upregulation of various modifying enzymes broadly predicts poor outcome in hepatocellular carcinoma.

[0220] Enumerated Aspects In some aspects, the present invention relates to the following non-limiting embodiments: Embodiment 1: An RNA molecule comprising at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme, wherein formation of said covalent bond inhibits said tRNA modifying enzyme. Embodiment 2: The RNA molecule of embodiment 1, wherein said RNA molecule is a tRNA molecule. Embodiment 3: The RNA molecule of embodiment 1 or 2, wherein said at least one nucleotide comprises an unnatural base. Embodiment 4: The RNA molecule of embodiment 3, wherein said at least one nucleotide is 5-halouridine (5-haloU), 5-halocytidine (5-haloC), 5-aza-cytidine (5-azaC), 8-haloadenosine (8-haloA), 8-azanebularine, 8-aza-adenosine (8-azaA), or 8-haloguanosine (8-haloG). Embodiment 5: The RNA molecule of any of embodiments 1 to 4, wherein the tRNA modifying enzyme is dihydrouridine synthase (DUS) or pseudouridine synthase (PUS). Embodiment 6: The tRNA modifying enzyme is selected from the group consisting of dihydrouridine synthase 1 (DUS1), dihydrouridine synthase 2 (DUS2), dihydrouridine synthase 3 (DUS3), dihydrouridine synthase 4 (DUS4), dihydrouridine synthase 1-like (DUS1L), dihydrouridine synthase 3-like (DUS3L), dihydrouridine synthase 4-like (DUS4L), pseudouridine synthase 1 (PUS1), pseudouridine synthase-like 1 (PUSL1), pseudouridine synthase 3 (PUS3), TruB pseudouridine synthase family member 1 (TRUB1), TruB pseudouridine synthase Family member 2 (TRUB2), dyskerin pseudouridine synthase 1 (DKC1), pseudouridine synthase 7 (PUS7), pseudouridine synthase 7-like (PUS7L), RNA pseudouridylate synthase domain-containing 1 (RPUSD1), RNA pseudouridylate synthase domain-containing 2 (RPUSD2), RNA pseudouridylate synthase domain-containing 4 (RPUSD4), pseudouridine synthase 10 (PUS10), tRNA methyltransferase 2 homolog A (TRMT2A), tRNA methyltransferase 2 homolog B (TRMT2B), NOP2 / Sun 6. The RNA molecule of any of embodiments 1 to 5, which is RNA methyltransferase 2 (NSUN2), NOP2 / Sun RNA methyltransferase 3 (NSUN3), NOP2 / Sun RNA methyltransferase 6 (NSUN6), DNA methyltransferase 2 (DNMT2), methyltransferase-like protein 1 (METTL1), WD repeat domain 4 (WDR4), adenosine deaminase TRNA-specific 1 (ADAT1), adenosine deaminase TRNA-specific 2 (ADAT2), adenosine deaminase TRNA-specific 2 (ADAT3), or ISCU. Embodiment 7: The unnatural base is 5-haloU and the tRNA modifying enzyme is DUS2, DUS1L, DUS3L, DUS4L, ISCU, PUS1, PUS3, PUS7, PUS10, PUSL1, PUS7L, RPUSD1, RPUSD2, RPUSD4, TRMT2A, TRMT2B, TRUB1, or TRUB2. the nucleotide containing an unnatural base is 5-azaC, and the tRNA modifying enzyme is DNMT2, NSUN2, NSUN3, or NSUN6; the nucleotide containing an unnatural base is 8-halo-G and the tRNA modifying enzyme is METTL1 or WDR4; or the nucleotide containing an unnatural base is 8-azaA, and the tRNA modifying enzyme is ADAT1, ADAT2, or ADAT3; The RNA molecule of any one of embodiments 1 to 6. Embodiment 8: The RNA molecule of any of embodiments 3 to 7, wherein the at least one nucleotide is located at a position corresponding to the natural position of a natural nucleotide in the natural tRNA that is modified by the tRNA modifying enzyme. Embodiment 9: The RNA molecule of any of embodiments 3 to 8, wherein the RNA molecule is a tRNA molecule, and the non-naturally modified base is 5-halouracil in the D-loop, t-psi-c loop, anticodon loop, or stem of the tRNA molecule. Embodiment 10: The RNA molecule of any of embodiments 1 to 9, comprising the sequence of any one of SEQ ID NOs: 1 to 53 or comprising at least about 80% sequence identity to the sequence of any one of SEQ ID NOs: 1 to 53. Embodiment 11: The RNA molecule of any of embodiments 1 to 10, which is an isolated tRNA molecule. Embodiment 12: The RNA molecule of any of embodiments 1 to 11, wherein the RNA molecule comprises two or more unnatural bases, and the two or more unnatural bases inhibit two or more different tRNA modifying enzymes. Embodiment 13: A composition comprising the RNA molecule of any of embodiments 1 to 12. Embodiment 14: The composition of embodiment 13, further comprising a pharmaceutically acceptable carrier, wherein said composition is a pharmaceutical composition. Embodiment 15: A method for killing a cell, comprising the steps of: A step of contacting the RNA molecule of any of aspects 1 to 12 with the tRNA modifying enzyme in a cell, wherein the formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cell. Embodiment 16: The method of embodiment 15, wherein said cell is a brain cancer cell, a gastrointestinal cancer cell, a kidney cancer cell, a liver cancer cell, or a lung cancer cell. Embodiment 17: The method of any of embodiments 15-16, wherein the cells are cancer cells in culture. Embodiment 18: A method for treating cancer in a subject in need thereof, comprising the steps of: administering to the subject an effective amount of the pharmaceutical composition of embodiment 14, wherein the RNA molecule contacts a tRNA modifying enzyme in cancer cells of the cancer, and formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cancer cells. Embodiment 19: The method of embodiment 18, wherein said cancer is lung cancer, brain cancer, gastrointestinal cancer, kidney cancer, or liver cancer. Embodiment 20: The method of embodiment 18, wherein said cancer is bladder cancer, breast cancer, cervical cancer, bile duct cancer, colon cancer, esophageal cancer, head / neck cancer, renal clear cell carcinoma, papillary renal carcinoma, liver cancer, non-small cell lung cancer, small cell lung cancer, prostate cancer, rectal cancer, sarcoma cancer, gastric cancer, uterine cancer, or a liquid tumor. Embodiment 21: The method of any of embodiments 18 to 20, further comprising administering chemotherapy to the subject.

[0221] Other Aspects The recitation of a list of elements in any definition of a variable herein includes the definition of that variable as any single element or combination (or sub-combination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment(s) or portion thereof.

[0222] The disclosures of all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with respect to certain embodiments, it will be apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. An RNA molecule comprising at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme, wherein the formation of the covalent bond inhibits the tRNA modifying enzyme.

2. The RNA molecule of claim 1, wherein the RNA molecule is a tRNA molecule.

3. 3. The RNA molecule of claim 1 or 2, wherein at least one nucleotide comprises an unnatural base.

4. 4. The RNA molecule of claim 3, wherein the at least one nucleotide is 5-halouridine (5-haloU), 5-halocytidine (5-haloC), 5-aza-cytidine (5-azaC), 8-haloadenosine (8-haloA), 8-azanebularine, 8-aza-adenosine (8-azaA), or 8-haloguanosine (8-haloG).

5. The RNA molecule of any one of claims 1 to 4, wherein the tRNA modifying enzyme is dihydrouridine synthase (DUS) or pseudouridine synthase (PUS).

6. The tRNA modifying enzyme is dihydrouridine synthase 1 (DUS1), dihydrouridine synthase 2 (DUS2), dihydrouridine synthase 3 (DUS3), dihydrouridine synthase 4 (DUS4), dihydrouridine synthase 1-like (DUS1L), dihydrouridine synthase 3-like (DUS3L), dihydrouridine synthase 4-like (DUS4L), pseudouridine synthase 1 (PUS1), pseudouridine synthase-like 1 (PUSL1), pseudouridine synthase 3 (PUS3), TruB pseudouridine synthase family member 1 (TRUB1), TruB pseudouridine synthase family member 2 (TRUB2), dyskerin pseudouridine synthase 1 (DKC1), pseudouridine synthase 7 (PUS7), pseudouridine synthase 7-like (PUS7L), RNA pseudouridylate synthase domain-containing 1 (RPUSD1), RNA pseudouridylate synthase domain-containing 2 (RPUSD2), RNA pseudouridylate synthase domain-containing 4 (RPUSD4), pseudouridine synthase 10 (PUS10), tRNA methyltransferase 2 homolog A (TRMT2A), tRNA methyltransferase 2 homolog B (TRMT2B), NOP2 / Sun 6. The RNA molecule of any one of claims 1 to 5, which is RNA methyltransferase 2 (NSUN2), NOP2 / Sun RNA methyltransferase 3 (NSUN3), NOP2 / Sun RNA methyltransferase 6 (NSUN6), DNA methyltransferase 2 (DNMT2), methyltransferase-like protein 1 (METTL1), WD repeat domain 4 (WDR4), adenosine deaminase TRNA-specific 1 (ADAT1), adenosine deaminase TRNA-specific 2 (ADAT2), adenosine deaminase TRNA-specific 2 (ADAT3), or ISCU.

7. the unnatural base is 5-haloU, and the tRNA modifying enzyme is DUS2, DUS1L, DUS3L, DUS4L, ISCU, PUS1, PUS3, PUS7, PUS10, PUSL1, PUS7L, RPUSD1, RPUSD2, RPUSD4, TRMT2A, TRMT2B, TRUB1, or TRUB2; the nucleotide containing an unnatural base is 5-azaC, and the tRNA modifying enzyme is DNMT2, NSUN2, NSUN3, or NSUN6; the nucleotide containing an unnatural base is 8-halo-G and the tRNA modifying enzyme is METTL1 or WDR4; or the nucleotide containing an unnatural base is 8-azaA, and the tRNA modifying enzyme is ADAT1, ADAT2, or ADAT3; An RNA molecule according to any one of claims 1 to 6.

8. 8. The RNA molecule of any one of claims 3 to 7, wherein the at least one nucleotide is at a position corresponding to a natural position of a natural nucleotide in a natural tRNA that is modified by the tRNA modifying enzyme.

9. The RNA molecule of any one of claims 3 to 8, wherein the RNA molecule is a tRNA molecule and the non-naturally modified base is 5-halouracil in the D-loop, t-psi-c loop, or anticodon loop of the tRNA molecule.

10. 10. The RNA molecule of any one of claims 1 to 9, comprising the sequence of any one of SEQ ID NOs: 1 to 53 or comprising at least about 80% sequence identity to the sequence of any one of SEQ ID NOs: 1 to 53.

11. 11. The RNA molecule of any one of claims 1 to 10, which is an isolated tRNA molecule.

12. 12. The RNA molecule of any one of claims 1 to 11, wherein the RNA molecule comprises two or more unnatural bases, wherein the two or more unnatural bases inhibit two or more different tRNA modifying enzymes.

13. A composition comprising the RNA molecule of any one of claims 1 to 12.

14. 14. The composition of claim 13, further comprising a pharmaceutically acceptable carrier, wherein the composition is a pharmaceutical composition.

15. A method for killing cells, comprising the steps of:

13. Contacting the RNA molecule of any one of claims 1 to 12 with the tRNA modifying enzyme in a cell, wherein formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cell.

16. 16. The method of claim 15, wherein the cell is a brain cancer cell, a gastrointestinal cancer cell, a kidney cancer cell, a liver cancer cell, or a lung cancer cell.

17. The method of any one of claims 15 to 16, wherein the cells are cancer cells in culture.

18. 1. A method for treating cancer in a subject in need thereof, comprising: Administering to the subject an effective amount of the pharmaceutical composition of claim 14, wherein the RNA molecule contacts a tRNA modifying enzyme in cancer cells of the cancer, and formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cancer cells.

19. 19. The method of claim 18, wherein the cancer is lung cancer, brain cancer, gastrointestinal cancer, kidney cancer, or liver cancer.

20. 19. The method of claim 18, wherein the cancer is bladder cancer, breast cancer, cervical cancer, bile duct cancer, colon cancer, esophageal cancer, head / neck cancer, kidney clear cancer, papillary renal cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, prostate cancer, rectal cancer, sarcoma cancer, gastric cancer, uterine cancer, or a liquid tumor.

21. 21. The method of any one of claims 18 to 20, further comprising administering chemotherapy to the subject.

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