WO2022047294A1 - Méthodes de thérapie de cancers liés à myc - Google Patents

Méthodes de thérapie de cancers liés à myc Download PDF

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WO2022047294A1
WO2022047294A1 PCT/US2021/048208 US2021048208W WO2022047294A1 WO 2022047294 A1 WO2022047294 A1 WO 2022047294A1 US 2021048208 W US2021048208 W US 2021048208W WO 2022047294 A1 WO2022047294 A1 WO 2022047294A1
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myc
ythdf2
rna
cells
cancer
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PCT/US2021/048208
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Eugene YEO
Jaclyn EINSTEIN
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The Regents Of The University Of California
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • the present disclosure relates to methods for Myc-dependent cancer therapy. More particularly, the present disclosure relates to methods of inhibiting the interaction between a RNA binding protein and a RNA.
  • TFs DNA-binding transcription factors
  • RBPs RNA-binding proteins
  • Somatic mutations can cause aberrant RBP expression in cancer cells, which can promote cell division, cell survival, metastasis, angiogenesis, and host immune evasion.
  • RBPs remain largely unexplored as drug targets because their systematic evaluation has been limited by the lack of sensitive and efficient assays for phenotypic interrogation of individual RBPs.
  • MYC C-MYC
  • RBPs RNA-binding proteins
  • YTHDF2 YTH N-6 Methyladenosine RNA binding protein 2
  • YTHDF2 selectively binds m 6 A modified sites on RNA and localizes target transcripts to mRNA decay sites for degradation by the CCR4-NOT deadenylase complex.
  • loss of YTHDF2 sensitizes acute myeloid leukemia (AML) cells to TNF-induced apoptosis
  • overexpression of YTHDF2 in hepatocellular carcinoma (HCC) represses cell proliferation and growth by destabilizing EGFR mRNA.
  • the direct YTHDF2 target RNAs have yet to be defined in the mammary epithelial or in human breast cancer.
  • PRSS23 Serine protease 23
  • EndoMT endothelial to mesenchymal transition
  • PRSS23 is not normally expressed in adult breast tissue, and its known functions include tissue remodeling in the developing fetus and in adult ovaries. PRSS23 has not been studied in the context of Myc-dependent cancers as described herein.
  • RNA binding protein binds to a RNA.
  • YTHDF2 YTH N-6 Methyladenosine RNA Binding Protein 2
  • YTHDF2 YTH N-6 Methyladenosine RNA Binding Protein 2
  • Also provided herein are in vitro methods of inducing apoptosis in a Myc-dependent cancer cell the method comprising administering a therapeutically effective amount of a nucleic acid targeting YTH N-6 Methyladenosine RNA Binding Protein 2 (YTHDF2), thereby inducing apoptosis in a Myc-dependent cancer cell.
  • YTHDF2 YTH N-6 Methyladenosine RNA Binding Protein 2
  • the method comprising administering to the triple negative breast cancer cells a therapeutically effective amount of a nucleic acid targeting YTH N-6 Methyladenosine RNA Binding Protein 2 (YTHDF2), thereby weakening triple negative breast cancer cells.
  • the nucleic acid comprises RNA, DNA, or combinations thereof.
  • the RNA comprises shRNA, miRNA, siRNA, or combinations thereof.
  • the RNA comprises shRNA.
  • the shRNA further comprises a lentiviral vector.
  • the lentiviral vector comprises The RNAi Consortium (TRC) lentiviral shRNA vector YTHDF2.
  • the shRNA further comprises an adenoviral vector (AAV).
  • the Myc-dependent cancer comprises ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, uterine cancer, leukemia, or combinations thereof.
  • the Myc-dependent cancer cell is a Myc- dependent breast cancer cell.
  • the administration of a nucleic acid targeting N-6 Methyladenosine RNA Binding Protein 2 induces unfolded protein accumulation in a Myc-dependent cancer cell. In some embodiments, the administration of a nucleic acid targeting N-6 Methyladenosine RNA Binding Protein 2 (YTHDF2) induces apoptosis in a Myc-dependent cancer cell.
  • FIGs. 1A-1 J show that RBP CRISPR screen identifies mRNA decay as a vulnerability in MYC-driven cancer.
  • FIG. 1A is an exemplary schematic describing the generation and implementation of the RBP-targeted CRISPR screen in MYC-ER HMECs.
  • FIG. IB shows cumulative distribution of normalized read counts for sgRNAs in each sample. Each condition is representative of two independent replicates. Two-sided Kolmogorov-Smirnov tests compared with day 0.
  • FIG. 1C shows comparison of score replicates on day 16. Inset highlights synthetic lethal candidate RBPs, and genes enriched for “negative regulation of macromolecule metabolic process” are labeled.
  • FIG. ID is an exemplary schematic describing the function of RBP candidates identified in FIG. 1C.
  • FIG. IE shows a boxplot comparing mRNA expression levels for MYC and YTHDF2 between TNBC and ER/PR/HER2 -positive tumors from TCGA data portal.
  • FIG. IF shows a western blot confirming shRNA-mediated knockdown in MYC-ER HMECs.
  • FIG. 1G shows quantification of Annexin V staining in MYC-ER HMECs with indicated knockdowns with and without 24 h of MYC induction.
  • FIG. 1H shows quantification of propidium iodide (PI) staining in MYC-ER HMECs with indicated knockdowns with and without 24 h of MYC induction.
  • FIG. II shows quantification of cell cycle phase using PI in MYC-ER HMECs with indicated knockdowns with and without 3 days of MYC induction.
  • FIG. 1J shows cell proliferation of shYTHDF2 cells compared with non-targeting control (NTC) over time in TNBC, HR+, and HER2+ breast cancer cell lines.
  • NTC non-targeting control
  • FIGs. 2A-2I show that depletion of YTHDF2 in TNBC cells suppresses tumor growth in vivo.
  • FIG. 2A shows an exemplary schematic of pooled shRNA screen conducted in MDA- MB-231-LM2 cells in vitro and in vivo.
  • DOX doxycycline
  • FIG. 2E shows average tumor volume over time of DOX-induced, YTHDF2-depleted xenografted mice compared with vehicle controls.
  • FIG. 2F shows images of mice 35 days following DOX induction compared with vehicle controls.
  • FIG. 2G shows tumor volumes 35 days following DOX induction compared with vehicle controls.
  • FIG. 2H shows qRT-PCR analysis relative to Gapdh of mRNA extracted from final tumors, 35 days following DOX induction.
  • FIG. 21 shows immunohistochemical staining of DOX-treated and vehicle control tumor sections, 35 days following DOX induction.
  • FIGs. 3A-3F show eCLIP and m 6 A-seq identify MAPK/ERK pathway transcripts that are regulated by YTHDF2.
  • FIG. 3A shows metagene profiles of filtered and unfiltered eCLIP and m 6 A-seq peak enrichment.
  • FIG. 3B shows motif analysis of unfiltered eCLIP (top) and m 6 A-seq (bottom) data. Representative of two independent replicates.
  • FIG. 3C shows Venn diagram overlaps of high-confidence m 6 A-seq peak and gene enrichment.
  • FIG. 3D shows four-way Venn diagram of overlapping, high-confidence YTHDF2 target genes among TNBC, HR+, and HER2+ breast cancer cell lines.
  • FIG. 3E shows a hierarchical cluster map illustrating expression levels of overlapping YTHDF2 target genes outlined in FIG. 3D. Gene clusters are depicted in the dendrogram.
  • FIG. 3F shows Gene Ontology (GO) enrichment of genes in dendrogram clusters from FIG. 3E.
  • FIGs. 4A-4H show depletion of YTHDF2 triggers activation of EMT.
  • FIG. 4B shows a Volcano plot describing the upregulated and downregulated genes in shYTHDF2 cells compared with NTC in MYC-induced HMECs.
  • FIG. 4C shows Gene Ontology (GO) enrichment of differentially expressed genes in shYTHDF2 cells compared with NTC in MYC-induced HMECs.
  • GO Gene Ontology
  • FIG. 4D shows immunofluorescent staining in TNBC cell lines. Arrowheads indicate cell projections.
  • FIG. 4E is an exemplary schematic displaying upstream signaling pathways that induce ERK1/2 signaling and EMT in breast cancer. Overlapping YTHDF2 targets in TNBC cell lines are listed.
  • FIG. 4F shows western blot analysis of cell lysates from NTC and shYTHDF2 TNBC, HR+, and HER2+ breast cancer cell lines of EMT transcription factor protein expression.
  • FIG. 4G shows western blot analysis of cell lysates from NTC and shYTHDF2 TNBC, HR+, and HER2+ breast cancer cell lines of ERK1/2 pathway activation and downstream effectors.
  • FIG. 4H shows qRT- PCR analysis relative to GAPDH of mRNA extracted from shYTHDF2 cells compared with NTC in TNBC, HR+, and HER2+ breast cancer cell lines.
  • FIGs. 5A-5I show that YTHDF2-depleted TNBC cells initiate apoptosis from intrinsic mitochondrial stress.
  • FIG. 5A shows and exemplary schematic describing the pathways that contribute to extrinsic versus intrinsic apoptosis.
  • FIG. 5B shows western blot analysis of cell lysates from non-targeting control (NTC) and shYTHDF2 TNBC, HR+, and HER2+ breast cancer cell lines assessing activation of the terminal unfolded protein response pathway.
  • FIG. 5C shows Genome Browser tracks (hgl9) depicting YTHDF2 eCLIP peaks over size- matched (SM) input and m 6 A methylation peaks over input on the PRSS23 transcript.
  • FIG. 5D shows boxplot displaying mRNA expression levels for MYC and PRSS23 in TNBC and ER/PR/HER2 -positive tumors from TCGA data portal.
  • FIG. 5E shows cell proliferation of shYTHDF2 and YTHDF2/PRSS23 co-depleted TNBC cells.
  • FIG. 5F shows western blot analysis of cell lysates from shYTHDF2 and YTHDF2/PRSS23 co-depleted TNBC cells assessing protein expression of UPR pathway genes and translation initiation factors.
  • FIG. 5G shows cumulative distribution of the fold change in mRNA expression of shYTHDF2 cells over NTC in MYC-induced HMECs. Describes direct TCF12 targets (ENCSR000BUN) or non-targets.
  • FIG. 5H shows Venn diagram describing the overlap of TCF12 targets determined in FIG. 5G with high-confidence YTHDF2 target genes and differentially upregulated genes in shYTHDF2 MYC-induced HMECs.
  • FIG. 51 shows quantification of reactive oxygen species (ROS) in shYTHDF2 and YTHDF2/PRSS23 co-depleted MDA-MB- 231-LM2 cells compared with NTC.
  • FIGs. 6A-6K show analysis of single cells within tumors reveals increased translation rates in YTHDF2-depleted, TurboRFP-tagged cells.
  • FIG. 6A is an exemplary schematic describing experimental workflow for generating cell lines, engrafting mice and dissociating tumors followed by single cell RNA-seq (scRNA) to profile the translatome within tumors.
  • FIG. 6B shows uniform manifold approximation and projection (UMAP) analysis of mRNA expression of merged cells from wild-type (WT) control-STAMP cells, WT RPS2-STAMP cells, shYTHDF2 control-STAMP cells, and shYTHDF2 RPS2-STAMP cells. Experiments performed in MDA-MB-231-LM2 cells.
  • FIG. 6C shows UMAP analysis of mRNA expression from merged cells from all samples. Outline indicates the cluster of highest TurboRFP expressing cells.
  • FIG. 6D shows UMAP analysis of mRNA expression from merged cells from all samples colored by expression score of differentially upregulated genes from in vitro bulk RNAseq in shYTHDF2 MYC-induced HMECs (FIG. 4B; upregulated). Outline indicates the cluster of highest TurboRFP-expressing cells.
  • FIG. 6E shows UMAP analysis of mRNA expression from merged cells. Outline indicates the cluster of highest TurboRFP expressing cells.
  • FIG. 6G shows clustered heatmaps of enriched Gene Ontology (GO) terms extracted from differentially expressed genes belonging to each mRNA expression Louvain cluster (p ⁇ 0.05).
  • FIG. 6H shows UMAP analysis of unfiltered EP KM of merged cells indicated by sample.
  • FIG. 61 shows UMAP analysis of unfiltered EPKM of merged cells indicated by average EPKM per cell.
  • FIG. 6J shows UMAP analysis of unfiltered EPKM of merged cells indicated by EPKM Louvain clustering. Cells filtered on the basis of assignment to control- or RPS2-STAMP cluster. Bar chart describes the fraction of each cluster contained in each sample.
  • FIG. 6K shows distribution of control-STAMP filtered, average EPKM per cell for TurboRFP low- and high-expressing populations.
  • FIGs. 7A-7F show that scRibo-STAMP identifies unique changes in the translatome of single cells clustering with TurboRFP-expressing populations.
  • FIG. 7A shows uniform manifold approximation and projection (UMAP) analysis of control-STAMP filtered EPKM of merged cells from RPS2-STAMP conditions indicated by mRNA expression Louvain clustering. Outline indicates the cluster of highest TurboRFP-expressing cells. Bar chart describes the fraction of each mRNA expression Louvain cluster in each EPKM Louvain cluster.
  • FIG. 7B shows UMAP analysis of control-STAMP filtered EPKM of merged cells from RPS2-STAMP samples indicated by EPKM Louvain clustering. Outline indicates the cluster of highest TurboRFP-expressing cells.
  • FIG. 7C shows clustered heatmaps of enriched Gene Ontology (GO) terms extracted from differentially edited genes from each EPKM Louvain cluster (p ⁇ 0.05).
  • FIG. 7E shows heatmap of normalized EPKM signatures for merged RPS2-STAMP cells for the top seven differentially edited genes per EPKM Louvain cluster.
  • FIG. 7F shows boxplot comparing protein expression levels for CST3, RPL24, and CFL1 between TNBC and ER/PR/HER2- positive tumors downloaded from TCGA’s Cancer Proteome Study of Breast Tissue.
  • FIGs. 8A-8B show that exemplary expression of PRSS23 in Myc-dependent cancer cells is lethal.
  • FIG. 8A is an exemplary schematic depicting function of YTHDF2 in control cells.
  • FIG. 8B is an exemplary schematic showing mechanism of programmed cell death with loss of YTHDF2 in Myc-dependent breast cancer cells.
  • FIG. 9 shows an exemplary schematic of a workflow of screening RBPs for therapeutic targets.
  • RNA-binding proteins RBPs
  • RBPs RNA-binding proteins
  • RNA binding protein RNA binding protein
  • a binding interaction between YTH N-6 Methyladenosine RNA Binding Protein 2 (YTHDF2) protein and Serine Protease 23 (PRSS23) transcript is present.
  • YTHDF2 binds to m 6 A modified sites on the 3’ untranslated region (UTR) of Prss23.
  • UTR untranslated region
  • abolishing the YTHDF2-PRSS23 interaction triggers apoptosis in Myc-dependent breast cancer cells.
  • the lethality of YTHDF2 knockout was identified by RBP CRISPR/Cas9 screening in isogenic Myc- inducible human mammary epithelial cells and the binding interaction with Prss23 was identified by overlapping YTHDF2 eCLIP-seq, m 6 A-seq, and RNA-seq datasets in several human Myc-dependent breast cancer cell lines.
  • the regulation of Prss23 mRNA stability is through reversible m 6 A RNA methylation.
  • Myc-dependent cancer cells maintain low to absent levels of PRSS23 transcript and protein through regulation by YTHDF2.
  • depletion of YTHDF2 increases translation of PRSS23 and triggers apoptosis in Myc-dependent cells.
  • PRSS23 transcript is lowly expressed in most adult tissues.
  • interruption of the binding interaction between YTHDF2 and Prss23 by depletion of YTHDF2 leads to upregulation of PRSS23 translation.
  • PRSS23 expression induces EMT-like phenotypes including, but not limited to upregulation of SNAIL expression, branching morphogenesis, cap-dependent translation and subsequent Myc expression.
  • Prss23 signals through the EIF2 pathway by upregulating translation initiation factors and effectively downregulating the PERK/eIF2a arm of the unfolded protein response (UPR).
  • unfolded proteins accumulate in the endoplasmic reticulum due to toxic levels of translation since amplified Myc expression routinely causes higher levels of transcription and translation.
  • ER-stress induced apoptosis is activated via the IREla/JNK arm of the UPR. In some embodiments, this ER-stress induced apoptosis is activated in Myc-dependent cells. In some embodiments, the YTHDF2 depletion leaves Myc-independent cells unaffected.
  • administration can refer to the administration of a composition to a subject or system to achieve delivery of the composition.
  • routes may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
  • administration may be ocular, oral, parenteral, or topical.
  • administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, or transdermal), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, or vitreal.
  • bronchial e.g., by bronchial instillation
  • buccal which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, or transdermal
  • enteral intra-arterial, intradermal, intragastric, intramedull
  • administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • an agent in general refers to any agent that elicits a desired pharmacological effect when administered to an organism.
  • an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population.
  • the appropriate population may be a population of model organisms.
  • an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc.
  • an agent is a substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • an agent has been or is required to be approved by a government agency before it can be marketed for administration to humans.
  • an agent can be an agent for which a medical prescription is required for administration to humans.
  • an agent does not require a medical prescription.
  • one or more agents can be used to elicit a desired pharmacological effect.
  • an agent includes a small molecule.
  • an agent includes a nucleic acid.
  • an agent comprises CRISPR/Cas9 components.
  • an agent comprises a single-guide RNA (sgRNA), wherein the sgRNA is targeted to an individual gene.
  • an agent comprises a short hairpin RNA (shRNA), wherein the shRNA is targeted to an individual gene.
  • a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.
  • a relevant cancer may be characterized by a solid tumor.
  • a relevant cancer may be characterized by a hematologic tumor.
  • hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like.
  • a cancer is a breast cancer.
  • a cancer is
  • the term “effective amount” or “therapeutically effective amount” means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition.
  • a therapeutically effective amount is one that reduces the incidence and/or severity of, stabilizes one or more characteristics of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
  • a therapeutically effective amount does not in fact require successful treatment be achieved in a particular individual.
  • a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.
  • term “therapeutically effective amount”, refers to an amount which, when administered to an individual in need thereof in the context of the inventive therapy, will block, stabilize, attenuate, or reverse a cancer-supportive process occurring in said individual, or will enhance or increase a cancer-suppressive process in said individual.
  • a “therapeutically effective amount” is an amount which, when administered to an individual diagnosed with a cancer, will prevent, stabilize, inhibit, or reduce the further development of cancer in the individual.
  • a particularly preferred “therapeutically effective amount” of a composition described herein reverses (in a therapeutic treatment) the development of a malignancy such as a pancreatic carcinoma or helps achieve or prolong remission of a malignancy.
  • a therapeutically effective amount administered to an individual to treat a cancer in that individual may be the same or different from a therapeutically effective amount administered to promote remission or inhibit metastasis.
  • the therapeutic methods described herein are not to be interpreted as, restricted to, or otherwise limited to a “cure” for cancer; rather the methods of treatment are directed to the use of the described compositions to “treat” a cancer, i.e., to effect a desirable or beneficial change in the health of an individual who has cancer.
  • Such benefits are recognized by skilled healthcare providers in the field of oncology and include, but are not limited to, a stabilization of patient condition, a decrease in tumor size (tumor regression), an improvement in vital functions (e.g., improved function of cancerous tissues or organs), a decrease or inhibition of further metastasis, a decrease in opportunistic infections, an increased survivability, a decrease in pain, improved motor function, improved cognitive function, improved feeling of energy (vitality, decreased malaise), improved feeling of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof.
  • a stabilization of patient condition e.g., a decrease in tumor size (tumor regression), an improvement in vital functions (e.g., improved function of cancerous tissues or organs), a decrease or inhibition of further metastasis, a decrease in opportunistic infections, an increased survivability, a decrease in pain, improved motor function, improved cognitive function, improved feeling of energy (vitality, decreased malaise), improved feeling of well-being,
  • regression of a particular tumor in an individual may also be assessed by taking samples of cancer cells from the site of a tumor such as a pancreatic adenocarcinoma (e.g., over the course of treatment) and testing the cancer cells for the level of metabolic and signaling markers to monitor the status of the cancer cells to verify at the molecular level the regression of the cancer cells to a less malignant phenotype.
  • a tumor such as a pancreatic adenocarcinoma
  • a therapeutically effective amount may be formulated and/or administered in a single dose.
  • a therapeutically effective amount may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
  • a subject and “patient” are used interchangeably throughout the specification to describe an organism, typically a mammal, human, or non-human, to whom treatment according to the methods of the present disclosure is provided.
  • Veterinary applications are contemplated by the present disclosure.
  • the terms include, but are not limited to, mammals, e.g., humans, other primates, pigs, hamsters, rats, mice, cows, horses, cats, dogs, sheep, and goats.
  • a subject is suffering from a relevant disease, disorder, or condition.
  • a subject displays one or more symptoms or characteristics of a disease, disorder, or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.
  • a subject is a patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • MYC is a transcription factor that can regulate the expression of up to 15% of genes in the genome.
  • c-myc is located on chromosome 8 and is believed to regulate expression of genes through binding on enhancer box sequences (E-boxes).
  • E-boxes enhancer box sequences
  • MYC The gene encoding MYC encodes the proto-oncogene, which is a nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis, and cellular transformation.
  • the encoded protein forms a heterodimer with the related transcription factor MAX.
  • This complex binds to the E box DNA consensus sequence and regulates the transcription of specific target genes. Amplification of this gene is frequently observed in numerous human cancers. Translocations involving this gene are associated with Burkitt’s lymphoma and multiple myeloma in human patients.
  • C-MYC (MYC) is the primary oncogenic driver of cancer gene expression programs in a broad spectrum of cancer types in which cells become “addicted” to and dependent on MYC for survival.
  • the MYC oncogene drives the pathogenesis of many hematopoietic malignancies, including Burkitt’s lymphoma (BL), Diffuse large B cell lymphoma (DLBCL), and Acute Lymphoblastic Leukemia (ALL).
  • BL Burkitt’s lymphoma
  • Diffuse large B cell lymphoma Diffuse large B cell lymphoma
  • ALL Acute Lymphoblastic Leukemia
  • a cancer is a hematologic cancer.
  • a hematologic cancer includes lymphomas (e.g. Non-Hodgkin lymphomas (NHL)), leukemias, and multiple myelomas.
  • a cancer is a Myc-dependent cancer.
  • a Myc-dependent cancer can include lymphoma, leukemia, osteosarcoma, hepatocellular carcinoma, squamous carcinoma, and pancreatic carcinoma.
  • a Myc-dependent cancer includes ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, uterine cancer, leukemia, or combinations thereof.
  • the Myc-dependent cancer is breast cancer.
  • RNA binding protein can refer to a protein that binds to the double or single stranded RNA in cells and participate in forming ribonucleoprotein complexes.
  • RNA binding proteins RBPs
  • RNA binding protein can refer to a protein that interacts with RNA molecules (e.g., mRNA) from synthesis to decay to affect their metabolism, localization, stability, and translation.
  • an RBP is a nuclear protein.
  • RBPs can include, but are not limited to, splicing factors, RNA stability factors, histone stem-loop binding proteins, or ribosomes.
  • a eukaryotic ribosome can include a collection of RBPs that can interact directly with mRNA coding sequences.
  • an RBP is a cytoplasmic protein.
  • an RNA binding protein comprises a ribosomal protein, wherein the ribosomal protein binds to a ribosome and an mRNA during translation. In some embodiments, an RNA binding protein comprises a ribosomal protein, wherein the ribosomal protein binds to a ribosome or an mRNA during translation.
  • the RNA binding protein comprises at least one of: SLTM, ZGPAT, PPARGC1B, PELP1, DCP2, CSTF3, TRA2B, ZNF638, SRSF9, LUC7L2, PTBP3, SF3B3, VCP, HNRNPA2B1, PTBP1, PCBP2, LSM14A, LSM12, DHX15, DDX27, DDX17, DDX21, IPO5, RPL22L1, RPL35, RPSA, MRPS34, NIFK, THUMPD1, RPUSD3, RRBP1, EEFSEC, UBAP2L, PUS7L, EIF4ENIF1, BICC1, EIF4E2, DARS2, TRDMT1, UPF3B, ZFP36L2, YTHDF2, EDC3, HNRNPR, UPF3A, ELAVL1, RBM27, XRN1, FUS, EXOSC7, PSPC1, CNOT7, CNOT6, CNOT
  • RNA-binding proteins have roles in controlling the fate of RNAs including the modulation of pre-mRNA splicing, RNA modification, translation, stability, and localization.
  • RBPs are a group of proteins that interact with RNA using an array of strategies from well-defined RNA-binding domains to disordered regions that recognize RNA sequence and/or secondary structures.
  • RNA-RBP complex can refer to a ribonucleoprotein complex comprising an RNA-binding protein (RBP) bound to a double or single stranded RNA in a cell.
  • an RNA-binding protein is YTH N-6 Methyladenosine RNA Binding Protein 2 (YTHDF2).
  • YTHDF2 YTH N-6 Methyladenosine RNA Binding Protein 2
  • the YTHDF2 binds to m 6 A modified sites.
  • the m 6 A modified sites are on the 3’ UTR of an RNA.
  • the m 6 A modified sites are on a PRSS23 transcript.
  • the m 6 A modified sites are on the 3’ UTR of a PRSS23 transcript.
  • YTH N6-methyladenosine RNA binding protein 2 is an RNA binding protein encoded by the YTHDF2 gene.
  • the YTHDF2 gene encodes a member of the YTH (YT521-B homology) superfamily containing YTH domain.
  • the YTH domain is usually located in the middle of the protein sequence and may function in binding to RNA.
  • this protein has a proline-rich region which may be involved in signal transduction, and an Alu-rich domain which is thought to be associated with human longevity.
  • reciprocal translocations between this gene and the Runxl (AML1) gene on chromosome 21 has been observed in patients with acute myeloid leukemia.
  • Methylation at the N6 position of adenosine is the most abundant RNA modification within protein-coding and long noncoding RNAs in eukaryotes and is a reversible process with important biological functions.
  • YTHDF proteins are the readers of m 6 A, the binding of which results in the alteration of the translation efficiency and stability of m 6 A-containing RNAs. It has been shown that disrupting YTHDF2-dependent mRNA degradation triggers apoptosis in triple-negative breast cancer (TNBC) cells and tumors (FIGs. 8A-8B).
  • crosslinking and immunoprecipitation is a method to identify RNA nucleotides that bind proteins of interest. CLIP typically delivers regions up to hundreds of nucleotides in length that are the approximate binding sites of a given protein.
  • Enhanced crosslinking and immunoprecipitation is a method to profile RNAs bound by an RNA binding protein of interest.
  • scRibo-STAMP profiling of translating mRNAs reveals unique alterations in the translatome of single cells within YTHDF2-depleted solid tumors, which selectively contribute to endoplasmic reticulum stress-induced apoptosis in triple-negative breast cancer (TNBC) cells.
  • STAMP APOBEC mediated profiling
  • STAMP is an integrated experimental and computational framework which demonstrates the discovery of RBP-RNA binding sites, including isoform-specific binding sites, at single-cell resolution.
  • STAMP is performed using with ribosome subunits.
  • ribosome-subunit STAMP refers to a chimeric protein, wherein the chimeric protein includes an RNA binding protein, and a ribosome subunit, wherein the ribosome subunit is fused to the RNA editing protein.
  • Ribo-STAMP refers to a chimeric protein, wherein the chimeric protein includes an RNA binding protein, and a ribosome subunit, wherein the ribosome subunit is fused to the RNA editing protein.
  • ribosome association and gene expression can be examined in a single-cell, in a homologous cell population, or in a heterologous cell population.
  • scRibo-STAMP refers to profiling of RBP-RNA interaction in single cells with the Ribo-STAMP method.
  • a critical role for YTHDF2 is in counteracting the global increase of mRNA synthesis in MYC-driven breast cancers.
  • nucleic acid is used to include any compound and/or substance that comprise a polymer of nucleotides.
  • a polymer of nucleotides are referred to as polynucleotides.
  • Exemplary nucleic acids or polynucleotides can include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization) or hybrids thereof.
  • RNAs ribonucleic acids
  • DNAs
  • Naturally- occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)).
  • a deoxyribose sugar e.g., found in deoxyribonucleic acid (DNA)
  • RNA ribonucleic acid
  • a nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art.
  • a deoxyribonucleic acid (DNA) can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and a ribonucleic acid (RNA) can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G).
  • RNA binding Protein 2 YTHDF2
  • the nucleic acid comprises RNA, DNA, or combinations thereof.
  • the nucleic acid is an RNA.
  • the RNA comprises shRNA, miRNA, siRNA, or combinations thereof.
  • the nucleic acid is a shRNA, wherein the shRNA induces knock down of a target RNA.
  • small hairpin RNAs refer to sequences of RNA, typically about 80 base pairs in length that include a region of internal hybridization that creates a hairpin structure.
  • ShRNA molecules are processed within the cell to form small interfering RNA (siRNA) which in turn knock down gene expression.
  • shRNA can be incorporated into plasmid vectors and integrated into genomic DNA for longer-term or stable expression, and thus longer knockdown of a target RNA.
  • an RNA can be a target RNA.
  • a target RNA refers to a RNA or RNA motif that is identified with a disease-related function.
  • various screening approaches and libraries can be used to identify drug-like small molecules with appropriate pharmacological properties.
  • a target RNA can be a therapeutically relevant target for a Myc-dependent cancer.
  • a target RNA can encode a RNA binding protein.
  • a target RNA is a RBP coding gene encoding the YTH N-6 Methyladenosine RNA Binding Protein 2 (YTHDF2).
  • the present disclosure provides methods of identifying RNA binding proteins (RBPs) implicated in Myc-driven breast cancer.
  • RBPs RNA binding proteins
  • the nucleic acid comprises CRISPR/Cas9 components.
  • the nucleic acid comprises a single guide RNA (sgRNA), wherein the sgRNA is targeted to an individual gene of a cell.
  • the sgRNA targets a RNA binding protein.
  • CRISPR/Cas9 refers to a technique of sequence specific genetic manipulation relying on the clustered regularly interspaced short palindromic repeats (CRISPR) pathway, which unlike RNA interference regulates gene expression at a transcriptional level.
  • CRISPR refers to a Cas-associated endonuclease and “CRISPR/Cas9” is a system that has been repurposed or engineered to target RNA instead of DNA in living cells, wherein the engineered Cas9 polypeptide-comprising nucleoprotein complex binds to RNA.
  • CRISPR/Cas9 can be used as a means to target RNA.
  • nucleoprotein complexes as provided herein comprise a Cas9 protein, a single guide RNA (sgRNA), and optionally an (chemically-modified or synthetic) antisense PAMmer oligonucleotide.
  • the PAMmer is an antisense oligonucleotide that serves to simulate a DNA substrate for recognition by Cas9 via hybridization to the target RNA.
  • gRNA or “guide RNA” as used herein refers to the guide RNA sequences used to target specific genes for correction employing the CRISPR technique.
  • Techniques of designing gRNAs and donor therapeutic polynucleotides for target specificity are well known in the art. For example, Doench, J., et al. Nature biotechnology 2014; 32(12): 1262-7 and Graham, D., et al. Genome Biol. 2015; 16: 260.
  • Single guide RNA or “sgRNA” is a specific type of gRNA that combines tracrRNA (transactivating RNA), which binds to Cas9 to activate the complex to create the necessary strand breaks, and crRNA (CRISPR RNA), comprising complimentary nucleotides to the tracrRNA, into a single RNA construct. Together, the Cas9 protein and sgRNA components allow recognition of hypothetically any RNA sequence.
  • the single guide RNA can recognize a target RNA, for example, by hybridizing to the target RNA.
  • the single guide RNA comprises a sequence that is complementary to the target RNA.
  • the sgRNA can include one or more modified nucleotides.
  • the sgRNA has a length that is about 10 nt (e.g., about 20 nt, about 30 nt, about 40 nt, about 50 nt, about 60 nt, about 70 nt, about 80 nt, about 90 nt, about 100 nt, about 120 nt, about 140 nt, about 160 nt, about 180 nt, about 200 nt, about 300 nt, about 400 nt, about 500 nt, about 600 nt, about 700 nt, about 800 nt, about 900 nt, about 1000 nt, or about 2000 nt).
  • nt e.g., about 20 nt, about 30 nt, about 40 nt, about 50 nt, about 60 nt, about 70 nt, about 80 nt, about 90 nt, about 100 nt, about 120 nt, about 140 nt, about 160 nt, about 180 nt, about 200
  • a single guide RNA can recognize a variety of RNA targets.
  • a target RNA can be messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (SRP RNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense RNA (aRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), retrotransposon RNA, viral genome RNA, or viral noncoding RNA.
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • SRP RNA signal recognition particle RNA
  • tRNA transfer RNA
  • tRNA transfer RNA
  • snRNA small nuclear RNA
  • snoRNA small nucleolar RNA
  • aRNA antisense RNA
  • IncRNA long noncoding RNA
  • miRNA
  • a target RNA can be an RNA involved in pathogenesis of conditions such as cancers, neurodegeneration, cutaneous conditions, endocrine conditions, intestinal diseases, infectious conditions, neurological conditions, liver diseases, heart disorders, or autoimmune diseases.
  • a target RNA can be a therapeutic target for conditions such as cancers, neurodegeneration, cutaneous conditions, endocrine conditions, intestinal diseases, infectious conditions, neurological conditions, liver diseases, heart disorders, or autoimmune diseases.
  • CRISPR/Cas9 systems are used to screen for RBP dependencies in Myc-dependent breast cancer.
  • the CRISPR/Cas9 systems include a sgRNA, wherein the sgRNA targets a Myc-dependent cancer-related gene encoding a RBP.
  • the sgRNA targets a RNA binding protein.
  • the sgRNA targets YTH N-6 Methyladenosine RNA Binding Protein 2 (YTHDF2) (FIG. 9).
  • a method of identifying RBPs in Myc-driven breast cancer can include using “enhanced crosslinking and immunoprecipitation (eCLIP)” as a method to profile RNAs bound by an RNA binding protein of interest.
  • the method can further include using Ribo-STAMP as a method to assess translation by directing C-to-U base editing of ribosome-bound transcripts using the cytidine deaminase enzyme APOBEC1 fused to the C-terminus of the 40S ribosomal protein S2 (RPS2).
  • Enhanced crosslinking and immunoprecipitation is a method to profile RNAs bound by an RNA binding protein of interest that showed thousand-fold improved recovery of protein-bound RNA. This technique can be modified and used to profile RNAs bound by specific ribosomal subunit proteins (Ribo-eCLIP).
  • enhanced crosslinking and immunoprecipitation recovers protein-coding mRNAs (with a particular enrichment for coding sequence regions), and the normalized Ribo-eCLIP enrichment correlates with translation rate estimates from independent approaches.
  • Ribo-eCLIP enables mapping translation rate from a variety of cell lines and tissue models.
  • STAMP Surveying targets by APOBEC mediated profiling
  • STAMP is a method of efficiently detecting RBP-RNA interaction, and identifying RBP- and cell type-specific RNA-protein interactions, by using a chimeric protein including an RNA binding protein and RNA editing protein.
  • STAMP is an integrated experimental and computational framework which demonstrates the discovery of RBP-RNA binding sites, including isoform-specific binding sites, at single-cell resolution.
  • STAMP is performed with computational methods that de-multiplex multiple RBPs by clustering cells using only edit signatures, allowing deconvolution of targets for multiplexed RBPs, and the cell-type specific binding of an RBP in a heterogeneous mixtures of cell-types.
  • STAMP allows for reproducible and quantitative identification of RBP-RNA binding sites, including isoform-specific binding sites. Further, STAMP can be used to determine a relative translation rate of an mRNA at single-cell resolution and in a heterogeneous mixture of cell-types. In some embodiments, STAMP can be used to examine translational landscapes at a single cell resolution. In some embodiments, STAMP can be used with specific ribosome subunits, wherein gene expression can be measured simultaneously with detection of ribosome association.
  • STAMP can identify cell-type specific RBP binding sites. In some embodiments, STAMP can identify multiple RBP binding sites for different RBPs in a single cell type. In some embodiments, STAMP can identify multiple RBP binding sites for different RBPs in multiple cell types. In some embodiments, STAMP can identify isoform- specific RBP binding sites. In some embodiments, STAMP and long-read sequencing can be used to identify isoform-specific RBP target sites. In some embodiments, STAMP can identify binding sites of full-length RBPs by C-to-U RNA editing. In some embodiments, STAMP can identify binding sites on single-stranded RNA targets.
  • STAMP provides a method for cell-type specific and multiplexed-RBP target identification in single cells.
  • STAMP can identify mammalian cell-type specific RBP binding sites.
  • STAMP can identify plant cell-type specific RBP binding sites.
  • STAMP can identify bacterial cell-type specific RBP binding site.
  • ribosome-subunit STAMP refers to a chimeric protein, wherein the chimeric protein includes an RNA binding protein, and a ribosome subunit, wherein the ribosome subunit is fused to the RNA editing protein.
  • Ribo-STAMP uses small ribosomal subunits to measure transcriptome-wide ribosome association in single cells.
  • Ribo-STAMP can be used to simultaneously measure ribosome association and gene expression.
  • Ribo-STAMP allows mRNA editing while identifying ribosome association with the mRNA, and also distinguishes genes with varying levels of ribosome occupancy.
  • Ribo-STAMP uses edited and non-edited reads to reflect ribosome-associated and input gene expression values simultaneously.
  • the simultaneous readouts can be used in complex and heterogeneous cellular or in vivo models to address cell identity or disease states.
  • YTHDF2 YTH N-6 Methyladenosine RNA Binding Protein 2
  • YTHDF2 YTH N-6 Methyladenosine RNA Binding Protein 2
  • Also provided herein are in vitro methods of inducing apoptosis in a Myc-dependent cancer cell the method comprising administering a therapeutically effective amount of a nucleic acid targeting YTH N-6 Methyladenosine RNA Binding Protein 2 (YTHDF2), thereby inducing apoptosis in a Myc-dependent cancer cell.
  • YTHDF2 YTH N-6 Methyladenosine RNA Binding Protein 2
  • Also provided herein are methods of weakening triple negative breast cancer cells comprising administering to the triple negative breast cancer cells a therapeutically effective amount of a nucleic acid targeting YTH N-6 Methyladenosine RNA Binding Protein 2 (YTHDF2), thereby weakening triple negative breast cancer cells.
  • YTHDF2 YTH N-6 Methyladenosine RNA Binding Protein 2
  • administering the nucleic acid can result in a decreased expression of an RNA binding protein.
  • the decreased expression of the RNA binding protein inhibits the RNA binding protein interaction with an RNA.
  • the RNA binding protein binds to a modified site on the RNA.
  • the modified site is an m 6 A modified site.
  • the modified site is located on a 3’ untranslated region (UTR) of the RNA.
  • the inhibition of the interaction between the RNA binding protein and the RNA results in apoptosis in Myc-dependent cancer cells.
  • the Myc-dependent cancer cell is a Myc-dependent breast cancer cell.
  • the decreased expression of the RNA binding protein increases expression of the RNA.
  • the increased expression of the RNA results in an increased number of unfolded proteins.
  • the determining step (a) includes performing a physical exam, a lab test, an imaging test, a biopsy, or any combination thereof.
  • a physical exam can include examining a patient for abnormalities, changes in skin color, or enlargement of an organ.
  • a lab test can include a urine test, or a blood test.
  • an imaging test can include a computerized tomography (CT) scan, a bone scan, a magnetic resonance imaging (MRI), a positron emission tomography (PET) scan, an ultrasound, or an x-ray.
  • a biopsy can include obtaining a sample of cells from a patient and imaging the cells to determine the presence or absence of cancerous cells.
  • the determining step (a) can include any of the methods disclosed herein and other methods as known in the art.
  • a CRISPR/Cas9 lentiviral library was used containing 10 single-guide RNAs (sgRNAs) for each of 1,078 RBPs, 628 sgRNAs targeting essential genes as positive controls, and 1,058 non-targeting sgRNAs as negative controls.
  • sgRNAs single-guide RNAs
  • the small library size and specific focus on RBPs provides higher confidence hits and therefore higher reproducibility than whole-genome approaches typically using more shallow coverage of 3 or 4 sgRNAs per gene.
  • High-throughput sequencing confirmed that the plasmid library maintained sgRNA coverage and that aliquots were tightly correlated (less than 0.065% of sgRNAs received undetectable normalized read counts.
  • MYC-ER HMECs were transduced with the CRISPR library in biological duplicate, selected for transduced cells with puromycin, treated half of the cells with tamoxifen (TAM) to induce MYC activity, and isolated genomic DNA (gDNA) 8 and 16 days after puromycin removal (FIG. 1A).
  • TAM tamoxifen
  • gDNA isolated genomic DNA 8 and 16 days after puromycin removal
  • HNRNPA2B1, PTBP1, SRSF9 genes that repress exon inclusion
  • ZGPAT, SLTM repress transcription
  • EIF4ENIF1, EIF4E2 translation initiation
  • MYC-amplified cells typically contain increased quantities of mRNA, caused in part by 30 untranslated region (UTR) shortening, RBP candidates that regulate RNA stability and turnover were further examined. It was found that the overall survival of patients with TNBC tumors containing above median MYC expression levels was significantly improved when tumors contained below median expression levels of mRNA decay factors AGO2, EXOSC7, FUS, YTHDF2, and ZFP36L2.
  • YTHDF2 mediates mRNA turnover by recruiting the CCR4-NOT deadenylase complex to initiate deadenylation and decay of m 6 A-containing transcripts before localizing with bound targets to processing (P) bodies for committed degradation.
  • P processing
  • the role of YTHDF2-mediated turnover of methylated RNA in cancer is not clear because of conflicting findings regarding its function.
  • the direct and functionally relevant YTHDF2 target RNAs have yet to be defined in the mammary epithelium or in human breast cancer.
  • TNBC cell lines MDA-MB-231 and secondary lung metastatic MDA-MB-231 -LM2
  • HR+ and HER2+ breast cancer cell lines MCF-7 and SKBR3, respectively
  • a YTHDF2-targeting hairpin that was significantly depleted in resected tumors and in cells cultured in vitro (p ⁇ 0.001) was identified, indicating a growth disadvantage in TNBC cells upon silencing of YTHDF2 (FIGs. 2B-2D)
  • YTHDF2 inhibition negatively affects tumor growth in vivo
  • stable MDA-MB-231-LM2 cells transduced with the DOX-inducible shRNA identified by the screen were generated. Following DOX treatment, it was confirmed that cells expressing the highest levels of the YTHDF2-targeting shRNA became depleted over time when cultured in vitro, suggesting death of cells with sufficient YTHDF2 depletion.
  • Ythdf2fl/fl mice were generated by crossing CAG-CreERT mice with previously generated Ythdf2fl/fl mice to expose any effects on the viability of healthy cells in other organs.
  • Systemic genetic depletion of Ythdf2 resulted in no gross physiological abnormalities or changes in body weight for at least 4 weeks following TAM administration, nor did it induce programmed cell death in female reproductive tissues known to rely on m 6 A regulation by YTHDF2. This suggests that inhibition of Ythdf2 in an intact organism has no adverse effects in non-cancerous somatic tissues and that YTHDF2 depletion safely and specifically inhibits growth of cells predisposed to MYC addiction.
  • Example 3 - YTHDF2 targets are enriched for genes regulating growth factor signaling
  • MY C-induced HMECs did not display robust differences in m 6 A-modified sites compared with uninduced HMECs in either direction, and neither did HR+ or HER2+ breast cancer cell lines compared with MDA-MB-231 cells, suggesting that MYC activity does not determine the m 6 A landscape in mammary tumors (FIG. 3C).
  • RNA expression data was analyzed for overlapping YTHDF2 targets in TNBC cell lines (FIG. 3D). Using hierarchical clustering of Z scores, two clusters were identified in which YTHDF2 targets are highly expressed in TNBC cells compared with HR+ and HER2+ cells (FIG. 3E).
  • clusters were enriched for genes that regulate wound healing, cell adhesion, ERK1/2 signaling, and EMT, while clusters including genes that are also highly expressed in MCF-7 cells generally lack enrichment for genes belonging to these ontology terms (FIG. 3F).
  • TNBC cells were verified to undergo expression changes resembling EMT following YTHDF2 depletion by performing RNA sequencing (RNA-seq) in YTHDF2- depleted, MYC-induced HMECs in biological duplicate. It was found that transcripts that are bound by YTHDF2 are upregulated compared with transcripts lacking binding sites (FIG. 4A), but inspection of the individual upregulated mRNAs revealed that >50% of upregulated transcripts were not direct YTHDF2 targets. These include CPA4, HM0X1, and MMP3, which are known to contribute to or are transcribed in response to cell migration, wound healing, and metastatic phenotypes (FIG. 4B).
  • RNA expression transcripts per million [TPM] % 1) in nontargeting control (NTC) cells yet were dramatically increased (-30-85 TPM) in YTHDF2-depleted cells.
  • TPM nontargeting control
  • results show that upregulated genes overlapping with those in YTHDF2-depleted MYC-induced HMECs over YTHDF2-depleted uninduced HMECs were enriched for ontologies associated with inflammatory and stress response, but not EMT-specific pathways, indicating that general inhibition of YTHDF2 promotes EMT and growth factor signaling, but only in conjunction with elevated MYC activity does YTHDF2 inhibition lead to apoptosis.
  • YTHDF2- depleted TNBC cells displayed a spindle-shape morphology denoted by high levels of vimentin localized to cell projections (FIG. 4D). Additionally, it was found that several upstream regulators of EMT are YTHDF2 targets (FIG. 4E) and that depletion of YTHDF2 resulted in upregulation of the downstream EMT TFs ZEB-1 and SNAIL (FIG. 4F). EMT is executed transcriptionally in response to the activation of the MAPK/ERK cascades, often leading to upregulation of MYC itself and increased expression of several translation initiation factors known to contribute to cancer progression.
  • MYC targets were bound by YTHDF2.
  • the adaptive endoplasmic reticulum stress pathway is typically engaged during EMT to alleviate metabolic and oxidative stress that accompanies cancer cell transformation and growth by facilitating protein folding and averting cell death.
  • activation of the adaptive unfolded protein response (UPR) was detected in all breast cancer subtypes, including upregulation of genes downstream of the serine/threonine
  • PERK kinase, ATF4, GADD34, and CHOP and splicing of XBP1 (sXBPl) downstream of the serine/threonine IRE1 kinase (FIG. 4H).
  • Example 5 Depletion of YTHDF2 sensitizes TNBC cells to proteotoxicity
  • MY C can actuate both cell proliferation and apoptosis
  • YTHDF2 has recently been shown to sensitize MYC-amplified acute myeloid leukemia (AML) cells to TNF-induced apoptosis, the extrinsic and intrinsic apoptotic pathways driving this phenotype were dissected (FIG. 5A).
  • TNF receptor superfamily member 10b (TNFRSF10B; i.e., DR5) was first observed by probing DR5-induced apoptotic activity through co-depletion of YTHDF2 and DR5.
  • TNFRSF10B TNF receptor superfamily member 10b
  • PRSS23 serine protease 23
  • Example 6 Depletion of YTHDF2 boosts translation rates in single cells across tumors
  • rates of protein synthesis in these cells was measured using puromycin incorporation as a surrogate measure of protein translation. It was observed that YTHDF2-depleted cells contained higher levels of puromycin incorporation into nascent peptides, indicating increased levels of protein synthesis.
  • Ribo-STAMP (surveying targets by APOBEC-mediated profiling) was used, a method to assess translation by directing C-to-U base editing of ribosome-bound transcripts using the cytidine deaminase enzyme APOBEC1 fused to the C-terminus of the 40S ribosomal protein S2 (RPS2).
  • a DOX-inducible, HA-tagged, RPS2- APOBEC1 (RPS2- STAMP), and APOBECl-only (control-STAMP) expressing cell lines both in wild-type (WT) and in DOX-inducible shRNA-targeting YTHDF2 (TurboRFP-tagged) MDA-MB-231- LM2 cells were generated. After engraftment, mice were given DOX water for 3 days to induce both shRNA and STAMP transgene expression prior to tumor resection, dissociation, and single-cell RNA-seq (scRNA-seq) using 10X Chromium Single Cell capture. At this early time point, cells harboring molecularly distinct translational signatures driven by both tumor heterogeneity and gene expression changes were uncovered reflected by C-to-U sequence changes in mRNA (FIG. 6A).
  • YTHDF2-depleted tumor cell populations (control-STAMP and RPS2-STAMP) contained cells with high raw read coverage in mitochondrial genes compared with WT samples, indicating higher fractions of apoptotic or lysed cells.
  • UMAP uniform manifold approximation and projection
  • cluster 3 may contain fewer TurboRFP Hlgh cells that either have yet to undergo cell-cycle arrest or have not undergone sufficient YTHDF2 depletion to initiate apoptosis, as the mRNA expression profile in cluster 3 more closely resembles that of cluster 4 (FIG. 6G), which contains cells expressing proliferative and tumorigenic markers.
  • Ribo-STAMP was expected to capture the full spectrum of translational states in tumors regardless of YTHDF2 modulation. Indeed, the distribution of EPKM values for filtered cells revealed a full range of edits throughout the TurboRFP Low population (FIG. 6K). Generally, more edits per gene in the YTHDF2-depleted, TurboRFP Hlgh population were found, suggesting increased rates of protein synthesis (FIG. 6K).
  • Example 7 - scRibo-STAMP identifies unique translation profiles for single YTHDF2- depleted cells within heterogeneous tumors
  • Tumors are composed of subpopulations of cells that differ at both the genomic and proteomic levels. Interestingly, it was found that in addition to increased rates of protein synthesis, TurboRFP Hlgh cells also contained edits on more genes per cell on average, suggesting that YTHDF2 depletion may initiate distinct translation programs. To identify differentially translated mRNAs, a subpopulation of cells containing the highest number of TurboRFP Hlgh cells with high EPKM was observed. This group comprised enrichment for cells assigned to cluster
  • FIG. 7A confirming that many of these cells originated from the shYTHDF2 RPS2- STAMP tumor.
  • Re-visualizing the UMAP space by Louvain clustering on EPKM produced very similar clusters with substantial overlap of mRNA expression-defined cluster 2 with EPKM-defined cluster A (FIGs. 7A and 7B). This supports the observation that modulation of m 6 A-modified transcripts on the RNA level incites widespread mRNA translation changes.
  • Clusters A and C contained the highest number of differentially edited transcripts (R99 genes) and enrichment for TurboRFP Hlgh cells (FIG.
  • cluster A (“pro-apoptotic” cluster) was enriched for edits within genes involved in apoptosis and antigen presentation
  • cluster C (“pro- tumorigenic” cluster) was enriched for edits within genes involved in preventing cell-cycle arrest, homotypic cell adhesion, and dampening oxidative stress, all of which are all associated with tumor progression (FIG. 7C).
  • CST3 cystatin C
  • CFL1 cofilin 1
  • RPL24 60S ribosomal protein L24
  • RPL24 is required for polysome assembly and is known to be upregulated in MYC-driven cancers
  • CFL1 is known to drive invasiveness in basal breast cancers.
  • TNBC tumors generally express significantly higher levels of CFL1 and RPL24 protein and less CST3 protein compared with HR+ and HER2+ tumors, supporting a role for CST3 in promoting MYC-driven apoptosis (FIG. 7F).
  • Ribo-STAMP data corroborated the findings that YTHDF2-depleted tumor cells contain unique translatomes displaying increased translation of tumorigenic and apoptotic transcripts while lacking translation of cell cycle regulators important for maintaining oncogenic proliferation.

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Abstract

Sont ici décrites des méthodes de thérapie de cancers liés à Myc, les méthodes comprenant l'administration d'un petit ARN en épingle à cheveux (sh-RNA) contenant un vecteur lentiviral, le sh-RNA étant dirigé vers une protéine de liaison à l'ARN identifiée. La protéine de liaison à l'ARN identifiée peut, dans certains cas, être la protéine 2 de liaison à l'ARN à modification YTH N-6 méthyladénosine 2 (YTHDF2).
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WO2021076617A1 (fr) * 2019-10-14 2021-04-22 The Regents Of The University Of California Composés anticancéreux à large spectre

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