Patent Application
20250025478
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jun. 27, 2024, is named 243735_000376_SL.xml and is 4,502,680 bytes in size.
This application relates to methods of inhibiting an inflammation- or hypoxia-induced death of a cell involving inhibiting an RNF213 signaling pathway. The application further relates to methods of preventing or treating various diseases and disorders (e.g., vascular occlusive events or disorders, autoimmune or inflammatory diseases, diseases associated with lipotoxicity, liver damage, cancer) in a subject involving administering to the subject an inhibitor of expression of RNF213 protein or an inhibitor of one or more functions of the RNF213 protein. The application still further relates to methods for enhancing the effectiveness of a cancer treatment in a subject where the cancer involves a hypoxic tumor microenvironment involving co-administering the cancer treatment with an inhibitor or activator of different functions of the RNF213 protein. The application also relates to methods of eliminating drug-resistant tumor cells (a.k.a. drug-tolerant persister cells, DTPs) by co-administering an inhibitor of the expression of RNF213 protein or by inhibiting or activating specific functions of the RNF213 protein.
Heart attack and stroke are the leading causes of death globally, often necessitating surgical intervention in addition to medications such as statins, thiazide diuretics, beta-blockers, and alpha-blockers to prevent further harm. Most drugs do not specifically target these conditions. Moyamoya disease (MMD) is a rare syndrome causing precocious strokes, often in teenagers and young adults, that are characterized by steno-occlusion of the carotid artery, a critical supplier of blood to the brain. MMD is also associated with more common types of steno-occlusive diseases and lipotoxicity. Currently, there is no effective medical treatment for MMD aside from complex surgical interventions. Single nucleotide polymorphisms (SNPs) in RNF213 are known to be associated with MMD. RNF213 is a giant (˜600 kDa) AAA-ATPase/ubiquitin ligase, which oligomerizes via its ATPase domains and has two potential E3 ubiquitin ligase domains (RING, RZ). RNF213 has been shown to protect the host against various pathogens through its oligomerized form and its RZ domain activity. Prior work has established that PTP1B negatively regulates the activity of RNF213 ubiquitin ligase activity and that loss of PTP1B regulation of RNF213 led to cell death in hypoxia. In particular PTP1B deficiency or inhibition can increase the death of HER2+ breast cancer cells maintained at ≤1% O2. Most of the highly penetrant MMD SNPs reside within the RING domain of RNF213.
In HER2+ breast cancer, a common problem with current established HER2+ breast cancer treatment regimens is the emergence of drug-tolerant persisters (DTPs), which can seed tumor recurrence.
As specified in the Background section above, there is a great need in the art for development of new therapeutic strategies for Moyamoya disease (MMD), as well as for patients with more conventional occlusive events (e.g., stroke, myocardial infarction, arterial thrombosis), diseases of lipotoxicity (e.g., hepatic steatosis), and cancer (e.g., HER2+ breast cancer). The present application addresses these and other needs.
In one aspect, provided herein is a method of inhibiting inflammatory cell death in a subject in need thereof, comprising inhibiting the RZ domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of inhibiting inflammatory cell death in a subject in need thereof, comprising inhibiting a functional ATPase domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of inhibiting inflammatory cell death in a subject in need thereof, comprising inhibiting RNF213 protein expression or degrading RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of inhibiting inflammatory cell death in a subject in need thereof, comprising activating the RING domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of inhibiting inflammatory cell death in a subject in need thereof, comprising inhibiting oligomerization of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of inhibiting inflammatory cell death in a subject in need thereof, comprising inhibiting an RNF213 activator in the cells of the subject.
In some embodiments of any of the above-described methods, the inflammatory cell death is pyroptotic cell death.
In another aspect, provided herein is a method of inhibiting hypoxia-induced cell death in a subject in need thereof, comprising inhibiting the RZ domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of inhibiting hypoxia-induced cell death in a subject in need thereof, comprising inhibiting a functional ATPase domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of inhibiting hypoxia-induced cell death in a subject in need thereof, comprising inhibiting RNF213 protein expression or degrading RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of inhibiting hypoxia-induced cell death in a subject in need thereof, comprising activating the RING domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of inhibiting hypoxia-induced cell death in a subject in need thereof, comprising inhibiting oligomerization of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of inhibiting hypoxia-induced cell death in a subject in need thereof, comprising inhibiting an RNF213 activator in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating a vascular occlusive event or disorder in a subject in need thereof, comprising inhibiting the RZ domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating a vascular occlusive event or disorder in a subject in need thereof, comprising inhibiting a functional ATPase domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating a vascular occlusive event or disorder in a subject in need thereof, comprising inhibiting RNF213 protein expression or degrading RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating a vascular occlusive event or disorder in a subject in need thereof, comprising activating the RING domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating a vascular occlusive event or disorder in a subject in need thereof, comprising inhibiting oligomerization of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating a vascular occlusive event or disorder in a subject in need thereof, comprising inhibiting an RNF213 activator in the cells of the subject.
In some embodiments of any of the above-described methods, the vascular occlusive event or disorder is a stroke, myocardial infarction, arterial thrombosis, atherosclerosis, peripheral arterial disease, renal vascular disease, Moyamoya disease (MMD), or Moyamoya syndrome.
In another aspect, provided herein is a method of preventing or treating an autoimmune or inflammatory disease in a subject in need thereof, comprising inhibiting the RZ domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating an autoimmune or inflammatory disease in a subject in need thereof, comprising inhibiting a functional ATPase domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating an autoimmune or inflammatory disease in a subject in need thereof, comprising inhibiting RNF213 protein expression or degrading RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating an autoimmune or inflammatory disease in a subject in need thereof, comprising activating the RING domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating an autoimmune or inflammatory disease in a subject in need thereof, comprising inhibiting oligomerization of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating an autoimmune or inflammatory disease in a subject in need thereof, comprising inhibiting an RNF213 activator in the cells of the subject.
In some embodiments of any of the above-described methods, the autoimmune or inflammatory disease is Graves' disease, systemic lupus erythematosus, Sjögren's syndrome, multiple sclerosis, or rheumatoid arthritis.
In another aspect, provided herein is a method of preventing or treating a disease associated with lipotoxicity in a subject in need thereof, comprising inhibiting the RZ domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating a disease associated with lipotoxicity in a subject in need thereof, comprising inhibiting a functional ATPase domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating a disease associated with lipotoxicity in a subject in need thereof, comprising activating the RING domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating a disease associated with lipotoxicity in a subject in need thereof, comprising inhibiting RNF213 protein expression or degrading RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating a disease associated with lipotoxicity in a subject in need thereof, comprising inhibiting oligomerization of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or treating a disease associated with lipotoxicity in a subject in need thereof, comprising inhibiting an RNF213 activator in the cells of the subject.
In some embodiments of any of the above-described methods, the disease associated with lipotoxicity is hepatic steatosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), primary lipodystrophy, or secondary lipodystrophy.
In another aspect, provided herein is a method of preventing or limiting liver damage in a subject having hepatic steatosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), primary lipodystrophy, or secondary lipodystrophy, the method comprising inhibiting the RZ domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or limiting liver damage in a subject having hepatic steatosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), primary lipodystrophy, or secondary lipodystrophy, the method comprising inhibiting a functional ATPase domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or limiting liver damage in a subject having hepatic steatosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), primary lipodystrophy, or secondary lipodystrophy, the method comprising activating the RING domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or limiting liver damage in a subject having hepatic steatosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), primary lipodystrophy, or secondary lipodystrophy, the method comprising inhibiting RNF213 protein expression or degrading RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or limiting liver damage in a subject having hepatic steatosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), primary lipodystrophy, or secondary lipodystrophy, the method comprising inhibiting oligomerization of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing or limiting liver damage in a subject having hepatic steatosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), primary lipodystrophy, or secondary lipodystrophy, the method comprising inhibiting an RNF213 activator in the cells of the subject.
In another aspect, provided herein is a method of preventing the generation of drug-tolerant persister cancer cells (DTPs) in a subject in need thereof who has been treated with a tyrosine kinase inhibitor (TKI), comprising inhibiting a functional ATPase domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing the generation of drug-tolerant persister cancer cells (DTPs) in a subject in need thereof who has been treated with a tyrosine kinase inhibitor (TKI), comprising inhibiting RNF213 protein expression or degrading RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing the generation of drug-tolerant persister cancer cells (DTPs) in a subject in need thereof who has been treated with a tyrosine kinase inhibitor (TKI), comprising inhibiting the RING domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing the generation of drug-tolerant persister cancer cells (DTPs) in a subject in need thereof who has been treated with a tyrosine kinase inhibitor (TKI), comprising inhibiting oligomerization of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of preventing the generation of drug-tolerant persister cancer cells (DTPs) in a subject in need thereof who has been treated with a tyrosine kinase inhibitor (TKI), comprising inhibiting an RNF213 activator in the cells of the subject.
In another aspect, provided herein is a method of sensitizing cancer cells to a tyrosine kinase inhibitor (TKI) in a subject in need thereof, comprising inhibiting a functional ATPase domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of sensitizing cancer cells to a tyrosine kinase inhibitor (TKI) in a subject in need thereof, comprising inhibiting RNF213 protein expression or degrading RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of sensitizing cancer cells to a tyrosine kinase inhibitor (TKI) in a subject in need thereof, comprising inhibiting the RING domain of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of sensitizing cancer cells to a tyrosine kinase inhibitor (TKI) in a subject in need thereof, comprising inhibiting oligomerization of RNF213 protein in the cells of the subject.
In another aspect, provided herein is a method of sensitizing cancer cells to a tyrosine kinase inhibitor (TKI) in a subject in need thereof, comprising inhibiting an RNF213 activator in the cells of the subject.
In another aspect, provided herein is a method of treating a cancer in a subject in need thereof or preventing recurrence of a cancer in a subject who has been treated with a targeted therapy, comprising activating the RZ domain of RNF213 protein in cancer cells of the subject.
In another aspect, provided herein is a method of treating a cancer in a subject in need thereof or preventing recurrence of a cancer in a subject who has been treated with a targeted therapy, comprising inhibiting a functional ATPase domain of RNF213 protein in cancer cells of the subject.
In another aspect, provided herein is a method of treating a cancer in a subject in need thereof or preventing recurrence of a cancer in a subject who has been treated with a a targeted therapy, comprising inhibiting the RING domain of RNF213 protein in cancer cells of the subject.
In another aspect, provided herein is a method of treating a cancer in a subject in need thereof or preventing recurrence of a cancer in a subject who has been treated with a targeted therapy, comprising inhibiting oligomerization of RNF213 protein in cancer cells of the subject.
In various embodiments, the tyrosine kinase inhibitor (TKI) is lapatinib, tucatinib, neratinib, afatinib, alectinib, axitinib, bosutinib, brigatinib, cabozantinib, ceritinib, crizotinib, dasatinib, entrectinib, erlotinib, gefitinib, imatinib, larotrectinib, lorlatinib, nilotinib, pazopanib, ponatinib, pyrotinib, regorafenib, sorafenib, sunitinib, or vandetanib, or a combination thereof. In some embodiments, the tyrosine kinase inhibitor (TKI) is lapatinib, tucatinib, or neratinib, or a combination thereof.
In various embodiments, the cells express human epidermal growth factor receptor 2 (HER2), HER4, epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), rearranged during transfection (RET), c-ros oncogene 1 (ROS1), mesenchymal-epithelial transition factor (MET), tropomyosin receptor kinase (TRK), breakpoint cluster region-abelson murine leukemia viral oncogene homolog 1 (BCR-ABL), anaplastic lymphoma kinase (ALK), KIT proto-oncogene, receptor tyrosine kinase (KIT), sarcoma (Src) oncogene, or rapidly accelerated fibrosarcoma (RAF), or a combination thereof.
In various embodiments, the cancer is a breast cancer, a pancreatic cancer, a gastric cancer, a cervical cancer, a colorectal cancer, a head and neck cancer, a thyroid cancer, a prostate cancer, an ovarian cancer, non-small cell lung cancer (NSCLC), endometrial cancer, bladder cancer, melanoma, renal cell carcinoma, hepatocellular carcinoma, seminoma, gastrointestinal stromal tumor (GIST), glioblastoma, inflammatory myofibroblastic tumor (IMT), dermatofibrosarcoma protuberans, chronic myeloid leukemia (CML), or acute lymphoblastic leukemia (ALL). In some embodiments, the breast cancer is a human epidermal growth factor receptor 2 (HER2+) breast cancer.
In some embodiments of any of the above-described methods, the RNF213 activator is ABL1/2 or TNK1. In some embodiments, inhibiting ABL1/2 comprises administering to the subject an effective amount of asciminib, dasatinib, imatinib, AP 24149, ponatinib, ruserontinib, PD173952, bosutinib, rebastinib, olverembatinib, KW-2449, bafetinib, or nilotinib. In some embodiments, inhibiting TNK1 comprises administering to the subject an effective amount of TP-5801, TP-5801 TFA, XMD8-92, CZC-25146, or CZC-25146 hydrochloride.
In some embodiments of any of the above-described methods, degrading RNF213 protein comprises administering to the subject an effective amount of PROTAC or a molecular glue.
In some embodiments of any of the above-described methods, inhibiting RNF213 protein expression comprises administering to the subject an effective amount of an siRNA, an shRNA, an anti-sense oligonucleotide, or a site-specific nuclease.
In another aspect, provided herein is a method of treating a cancer in a subject in need thereof or preventing recurrence of a cancer in a subject who has been treated with a chemotherapy, radiotherapy, or a targeted therapy, comprising activating the RZ domain of RNF213 protein in cancer cells of the subject.
In another aspect, provided herein is a method of treating a cancer in a subject in need thereof or preventing recurrence of a cancer in a subject who has been treated with a chemotherapy, radiotherapy, or a targeted therapy, comprising activating a functional ATPase domain of RNF213 protein in cancer cells of the subject.
In another aspect, provided herein is a method of treating a cancer in a subject in need thereof or preventing recurrence of a cancer in a subject who has been treated with a chemotherapy, radiotherapy, or a targeted therapy, comprising inhibiting the RING domain of RNF213 protein in cancer cells of the subject.
In another aspect, provided herein is a method of treating a cancer in a subject in need thereof or preventing recurrence of a cancer in a subject who has been treated with a chemotherapy, radiotherapy, or a targeted therapy, comprising promoting oligomerization of RNF213 protein in cancer cells of the subject.
In another aspect, provided herein is a method for eliminating hypoxic tumor cells in a subject in need thereof, comprising activating the RZ domain of RNF213 protein in the tumor cells of the subject.
In another aspect, provided herein is a method for eliminating hypoxic tumor cells in a subject in need thereof, comprising activating a functional ATPase domain of RNF213 protein in the tumor cells of the subject.
In another aspect, provided herein is a method for eliminating hypoxic tumor cells in a subject in need thereof, comprising inhibiting the RING domain of RNF213 protein in the tumor cells of the subject.
In another aspect, provided herein is a method for eliminating hypoxic tumor cells in a subject in need thereof, comprising promoting oligomerization of RNF213 protein in the tumor cells of the subject.
In another aspect, provided herein is a method for enhancing effectiveness of an anti-cancer treatment in a subject in need thereof, comprising co-administering the anti-cancer treatment with an effective amount of an activator of the RZ domain of RNF213 protein. In some embodiments, co-administering comprises administering to the subject the anti-cancer treatment and an activator of the RZ domain of RNF213 protein simultaneously in one composition or in separate compositions. In some embodiments, co-administering comprises administering the anti-cancer treatment and an activator of the RZ domain of RNF213 protein sequentially, in any order.
In another aspect, provided herein is a method for enhancing effectiveness of an anti-cancer treatment in a subject in need thereof, comprising co-administering the anti-cancer treatment with an effective amount of an activator of a functional ATPase domain of RNF213 protein. In some embodiments, co-administering comprises administering the anti-cancer treatment and the activator of the functional ATPase domain of RNF213 protein simultaneously in one composition or in separate compositions. In some embodiments, co-administering comprises administering the anti-cancer treatment and the activator of the functional ATPase domain of RNF213 protein sequentially, in any order.
In another aspect, provided herein is a method for enhancing effectiveness of an anti-cancer treatment in a subject in need thereof, comprising co-administering the anti-cancer treatment with an effective amount of an inhibitor of the RING domain of RNF213 protein. In some embodiments, co-administering comprises administering the anti-cancer treatment and an inhibitor of the RING domain of RNF213 protein simultaneously in one composition or in separate compositions. In some embodiments, co-administering comprises administering the anti-cancer treatment and an inhibitor of the RING domain of RNF213 protein sequentially, in any order.
In another aspect, provided herein is a method for enhancing effectiveness of an anti-cancer treatment in a subject in need thereof, comprising co-administering the anti-cancer treatment with an effective amount of a compound promoting oligomerization of RNF213 protein in cancer cells of the subject. In some embodiments, co-administering comprises administering the anti-cancer treatment and the compound promoting oligomerization of RNF213 protein simultaneously in one composition or in separate compositions. In some embodiments, co-administering comprises administering the anti-cancer treatment and the compound promoting oligomerization of RNF213 protein sequentially, in any order.
In some embodiments of any of the above-described methods, the anti-cancer treatment is a chemotherapy, targeted therapy, or antibody-drug conjugate (ADC) treatment.
In some embodiments of any of the above-described methods, the cancer is a breast cancer, pancreatic cancer, or cervical cancer. In some embodiments, the breast cancer is a HER2+ breast cancer.
In some embodiments of any of the above-described methods, the cancer has significant regions of hypoxia.
In some embodiments of any of the above-described methods, the functional ATPase domain of RNF213 protein is the 3rd or 4th ATPase domain.
In some embodiments of any of the above-described methods, an activator of a functional ATPase domain is a PTP1B inhibitor.
In some embodiments of any of the above-described methods, the subject is human.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
To facilitate an understanding of the principles and features of the various embodiments of the invention, various illustrative embodiments are explained below. Although exemplary embodiments of the invention are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the invention is limited in its scope to the details of construction and arrangement of components set forth in the following description or examples. The invention is capable of other embodiments and of being practiced or carried out in various ways. Also, in describing the exemplary embodiments, specific terminology will be resorted to for the sake of clarity.
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.
The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
The terms “inhibit” or “inhibition” as used herein refer to reducing a function or activity to an extent sufficient to achieve a desired biological or physiological effect. Inhibition may be complete or partial.
The phrase “pharmaceutically acceptable”, as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
As used herein, “pharmaceutically acceptable carrier” or “pharmaceutical acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).
The term “treating”, as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, delaying the progression of, delaying the onset of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term “treatment”, as used herein, unless otherwise indicated, refers to the act of treating as “treating” is defined immediately above. The term “treating” also includes adjuvant and neo-adjuvant treatment of a subject. For the avoidance of doubt, reference herein to “treatment” includes reference to curative, palliative and prophylactic treatment.
The phrase “effective amount” or “therapeutically effective amount” as used herein refers to an amount necessary (at dosages and for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount is at least the minimal amount, but less than a toxic amount, of an active agent which is necessary to impart therapeutic benefit to a subject.
The terms “patient”, “individual”, “subject”, and “animal” are used interchangeably herein and refer to mammals, including, without limitation, human and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.
“Drug-tolerant persister cells” or “drug-tolerant persister cancer cells” or “DTPs”, or the like, as used herein refer to a sub-population of cancer cells that survive under anti-cancer treatment(s), e.g., chemotherapy, radiation therapy, and/or targeted therapy.
It must also be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the context clearly dictates otherwise. For example, reference to a component is intended also to include composition of a plurality of components. References to a composition containing “a” constituent is intended to include other constituents in addition to the one named. In other words, the terms “a,” “an,” and “the” do not denote a limitation of quantity, but rather denote the presence of “at least one” of the referenced item.
Also, in describing the exemplary embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.
It is also to be understood that the mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a composition does not preclude the presence of additional components than those expressly identified.
The materials described hereinafter as making up the various elements of the present invention are intended to be illustrative and not restrictive. Many suitable materials that would perform the same or a similar function as the materials described herein are intended to be embraced within the scope of the invention. Such other materials not described herein can include, but are not limited to, materials that are developed after the time of the development of the invention, for example. Any dimensions listed in the various drawings are for illustrative purposes only and are not intended to be limiting. Other dimensions and proportions are contemplated and intended to be included within the scope of the invention.
Most tumors have hypoxic or anoxic areas1, and cancer cells adapt to limited oxygen in the tumor microenvironment (TME) by successively activating several signaling pathways. In normoxia, the transcription factors HIF1α and HIF2α (hereafter “HIF”) are hydroxylated by oxygen-dependent prolyl hydroxylases (PHD 1-3), bound by the Van Hippel Lindau tumor suppressor gene product (VHL), and targeted for ubiquitylation and proteasomal degradation2. PHD enzymes are inhibited in low oxygen, resulting in HIF stabilization and induction of hypoxia response genes. More severe hypoxia (≤1% O2) triggers the AMP-dependent kinase (AMPK) and endoplasmic reticulum (ER) stress pathways, which evoke distinct adaptive responses3,4. The activity of NF-κB, a key transcription factor controlling inflammation and innate immunity, also increases in hypoxia5 via (an) incompletely defined mechanism(s) that include(s) decreased PHD regulation of NF-κB pathway signaling components. Conversely, NF-κB is required for basal HIF expression, and thus directly and indirectly regulates the transcriptome of hypoxic cells6. The complex interplay between HIF-induced, metabolic (AMPK), integrated stress response (ER stress), and inflammatory/innate immune7,8(NF-κB) pathways helps determine cancer cell sensitivity to conventional (chemoradiation) and targeted therapy3, as well as whether tumor cell death evoked by antineoplastic therapies is “sterile” or immunogenic9-11.
The protein-tyrosine phosphatase PTP1B (encoded by PTPN1) resides on the cytosolic surface of the endoplasmic reticulum (ER), where it dephosphorylates incoming receptor tyrosine kinases (RTKs) and cytokine receptors12,13. Best known as a negative regulator of insulin and leptin signaling14-17, PTP1B is also required for Neu (encoding rodent HER2)-induced breast cancer in mice18,19. Moreover, PTPN1 is amplified (˜5%) and overexpressed (˜72%) in human cancer20,21. It was previously reported that PTP1B deficiency or inhibition increased the death of HER2+ breast cancer cells maintained at ≤1% O2 without altering the HIF, ER stress, or AMPK pathways22. Instead, hypoxia hypersensitivity required RNF213, a large (˜600 kDa) protein with AAA-ATPase and E3 ligase domains. PTP1B deficiency/inhibition promoted an increase in RNF213-dependent global ubiquitylation, and RNF213 was tyrosine phosphorylated and appeared to be a PTP1B substrate. However, key details, including exactly how PTP1B regulates RNF213 and the mechanism and type of RNF213-dependent cell death, remained unclear.
Single nucleotide polymorphisms (SNPs) in RNF213 are associated with Moyamoya disease (MMD), a rare syndrome characterized by precocious carotid artery stenosis and stroke, often in teenagers or young adults23-25. RNF213 features six AAA-ATPase modules, two of which are active, a RING domain, a recently discovered “RZ” E3 ligase domain, and large unstructured regions26-30. In an AAA-ATPase- and ISG15-dependent manner, RNF213 can oligomerize into a hexameric, ˜3.6 mDa complex26,28. RNF213 also localizes on lipid droplets31 (also dependent on AAA-ATPase activity/ISG15), and is required for lysophosphatidic acid (LPA)-induced cell death in vitro32. Transfection studies implicate RNF213 in NF-κB signaling30,32, and recently, the RNF213 RZ domain was found to ubiquitylate lipid A of Salmonella29. In concert, these findings suggest roles for RNF213 in lipotoxicity and inflammation, both potentially relevant for MMD pathogenesis. The most highly penetrant MMD SNPs affect the RNF213 RING, not the RZ domain27,30,33, leaving the detailed molecular pathogenesis of this disease—and its relationship, if any, to hypoxia hypersensitivity in PTP1B-deficient/inhibited breast cancer cells obscure.
In certain embodiments, inhibitors or activators of specific domains of the RNF213 protein may provide a therapeutic benefit to patients having one or more of breast cancer, pancreatic cancer, or other cancers with hypoxic tumor microenvironments (TMEs); to cancer patients treated with chemotherapy or targeted therapy that leave “drug-tolerant persister” cancer cells (DTPs), to individuals with Moyamoya Disease (MMD)/stroke/arthrosclerosis); non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), or other types of lipotoxicity (e.g., primary lipodystrophies); autoimmune diseases and inflammatory diseases.
In certain embodiments, inhibitors or activators of specific domains of the RNF213 protein may provide a therapeutic benefit, e.g., in combination with a HER2 tyrosine kinase inhibitor (TKI) to treat a drug-tolerant population (DTP) in breast cancer and potentially other malignancies. In some embodiments, inhibiting expression or promoting degradation of RNF213 can have salutary effects on preventing DTP formation.
In some embodiments, an RNF213-targeted agent can be used to treat the above-listed conditions. For instance, for hypoxic breast tumors (and other hypoxic tumors), agents can include small molecule activators of RNF213 ATPase activity, RNF213 oligomerization, or the RNF213 RZ domain, or small molecule inhibitors of the RNF213 RING domain. In some embodiments, small molecule activators or inhibitors may be used in combination with conventional chemotherapy, antibody drug conjugates (ADCs), targeted therapies, therapeutic antibodies, or combinations thereof.
In another embodiment, for Moyamoya disease (MMD), stroke, or atherosclerosis, inhibitors of the ATPase or RZ domain of RNF213 or RNF213 proteolysis targeting chimeras (PROTACs) may have therapeutic potential in MMD. Inhibitors of specific tyrosine kinases (for ABL1/2-asciminib, dasatinib, imatinib, nilotinib; for TNK1, TP-5801) that promote RNF213 oligomerization can also have therapeutic potential. RNF213 interactors identified by TurboID proximity-based labeling described herein are involved in stroke, atherosclerosis, lipotoxicity, and cholesterol metabolism. A hallmark of strokes can be inflammatory cell death (pyroptosis). RNF213 triggers pyroptosis through activation of NF-κB in a stress condition when accompanied by hypoxia. Therefore, ABL or TNK inhibitors, inhibitors of the RNF213 ATPase domain or RZ domain, inhibitors of RNF213 oligomerization, RNF213 PROTACs, or activators of RNF213 RING domain could have utility in limiting neuronal cell death in strokes, MI, and other steno-occlusive disorders.
In yet another embodiment, for non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), or lipotoxicity, RNF213 ATPase or RZ inhibitors, inhibitors of RNF213 oligomerization, RNF213 PROTACs, or activators of RNF213 RING domain may prevent or limit liver damage in NASH.
In certain embodiments, for autoimmune diseases and inflammatory diseases, wild-type RNF213 can promote NF-κB signaling through the degradation of CYLD/SPATA2. Impairing RNF213 ATPase activity or RZ activity, inhibiting RNF213 oligomerization, activators of RNF213 RING domain or inhibitors of tyrosine kinases for RNF213 (e.g., ABL1/2 or TNK1) could be therapeutic for such cases.
In another aspect, the present disclosure provides methods for preventing the generation of drug-tolerant persisters (DTPs), for example, in HER2+ breast cancer. RNF213 knockout cells are more susceptible to prescribed HER2+BC treatment drugs like lapatinib, tucatinib (HER2 inhibitors) suggesting that RNF213-KO cells are less likely to form DTPs. Combining a HER2-targeted agent with RNF213 PROTACs or inhibitors of other RNF213 domains could augment HER2-based therapies. ABL1/2 or TNK1 inhibition might also have benefits. In certain embodiments, for HER2+ breast cancer, combining a HER2-targeted agent with, e.g., RNF213 PROTACs, inhibiting RNF123 expression, or inhibitors or activators of specific RNF213 domains may be useful in the treatment of such cancers.
A tyrosine phosphorylation site in RNF213 was identified herein as being regulated by PTP1B (Y1275) and phosphorylated by ABL1/2. Y1275 phosphorylation promotes AAA-ATPase domain-dependent oligomerization of RNF213 and activation of its ubiquitin ligase activity. Proximity ligation experiments with Turbo-ID described herein identified multiple novel interacting proteins, including CYLD/SPATA2, a major negative regulator of NF-κB. Detailed analysis described herein shows that the RNF213, via its RZ domain, ubiquitylates CYLD/SPATA2 and, in concert with the LUBAC complex, leads to their degradation in hypoxia, NF-κB activation, induction of NLRP3, and inflammasome priming. The RZ domain also ubiquitylates Lipid A of Salmonella LPS, and in concert with LUBAC, promotes bacterial clearance by autophagy. Hypoxia-evoked ER stress then provides the “second signal” leading to inflammasome activation and Gasdermin D-dependent pyroptotic cell death. Hence, PTP1B inhibition/degradation, in addition to killing hypoxic tumor cells that are typically resistant to chemoradiation and targeted therapies, could “turn cold tumors hot” and facilitate immunological clearance. HER2+ breast cancer and many other breast cancer lines also show hypoxia hypersensitivity upon PTP1B inhibition. The RING domain of RNF213 negatively regulates RZ domain ubiquitin ligase activity and removes the requirement for the LUBAC complex to degrade CYLD/SPATA2 and activate NF-κB. The most common MMD SNPs affect the RING, and MMD alleles activate NF-κB. This may be related to the frequent co-occurrence of autoimmune and inflammatory diseases with MMD. Furthermore, several other interactors in the Turbo screen described herein are implicated in hypoxia and atherosclerosis such that RNF213 might be targeted for therapeutic benefit in those disorders.
As described herein, RNF213 promotes pyroptosis, a characteristic feature of vascular occlusive events. By understanding that tyrosine kinases ABL1/2 and TNK1 enhance RNF213 pyroptotic activity, use of tyrosine kinase inhibitors is explored for stroke treatment. Moreover, direct inhibitors targeting RNF213 are developed for greater specificity. A pathway and mechanism for targeting RNF213, a giant AAA-ATPase/ring finger-containing ubiquitin ligase is described, which promotes pyroptotic cell death in severe hypoxia. The role of RNF213 can require its RZ domain as well as its ATPase domain, whereas the RING domain inhibits the RZ domain. The ATPase activity of RNF213 controls oligomerization of RNF213 and is negatively regulated by the protein-tyrosine phosphatase PTP1B and positively regulated by ABL family kinases and TNK1. Single nucleotide polymorphism (SNPs) in RNF213 are known to be associated with Moyamoya disease. Inactivation of the RNF213 RING domain (where most of the highly penetrant MMD SNPs reside) promotes its RZ activity. Since this RZ activity is requires ATPase activity, the findings that RNF213 oligomerization is promoted by ABL family kinases and TNK1 could have therapeutic potential for Moyamoya disease, as well as for patients with more conventional occlusive events (e.g., stroke, myocardial infarction (MI), arterial thrombosis) and diseases of lipotoxicity (e.g., hepatic steatosis, lipodystrophy). Specifically, ABL, TNK1, or RNF213 RZ inhibitors or degraders could be active in these settings.
It was found that ABL1/2 phosphorylate, whereas PTP1B dephosphorylates, RNF213 on tyrosine 1275 (Y1275). Phosphorylation of this site promotes RNF213 oligomerization and activation of its E3 ligase activity. Via proximity-based proteomics, the CYLD-SPATA2 complex, a major negative regulator of NF-κB activation, was identified as an RNF213 substrate critical for hypoxia-induced cell death. Moreover, the RZ domain is required for CYLD-SPATA2 ubiquitylation/degradation, while the RING domain restrains RZ activity. Wild type RNF213 requires the LUBAC complex for effective CYLD-SPATA2 degradation, but RNF213 RING mutants, including highly penetrant SNPs associated with MMD, bypass this requirement. Increased RNF213 activity, caused by PTP1B deficiency/inhibition or RNF213 RING mutations, drives CYLD/SPATA2 ubiquitylation and degradation and increases NF-κB activity, priming cells for pyroptosis via the NLRP3 inflammasome. Concomitantly, hypoxia-induced ER stress delivers the “second signal” resulting in increased cancer cell death. The results described herein have implications for the mechanism of action and potential uses of dual inhibitors of PTP1B/TC-PTP (PTPN2)34, now in clinical trials for various cancers, as well as for the pathogenesis of MMD and other steno-occlusive disorders.
In some embodiments, the inhibitor of RNF213 protein expression is an siRNA, an shRNA, an antisense oligonucleotide, a miRNA, or a site-specific nuclease. Non-limiting examples of the inhibitor are described below.
In some embodiments, the inhibitor is a small interfering RNAs (siRNA), also known as short interfering RNA or silencing RNA. siRNAs are a class of double-stranded RNA molecules, typically about 20-25 base pairs in length that target nucleic acids (e.g., mRNAs) for degradation via the RNA interference (RNAi) pathway in cells. Such siRNA molecules typically include a region of sufficient homology to the target region, and are of sufficient length in terms of nucleotides, such that the siRNA molecules down-regulate target nucleic acid. It is not necessary that there be perfect complementarity between the siRNA molecule and the target, but the correspondence must be sufficient to enable the siRNA molecule to direct sequence-specific silencing, such as by RNAi cleavage of the target RNA. In some embodiments, the sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double-strand character of the molecule.
Specificity of siRNA molecules may be measured via the binding of the antisense strand of the molecule to its target RNA. Effective siRNA molecules are often fewer than 30 to 35 base pairs in length, e.g., to prevent stimulation of non-specific RNA interference pathways in the cell by way of the interferon response, however longer siRNA may also be effective. In various embodiments, the siRNA molecules are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length. In various embodiments, the siRNA molecules are about 35 to about 70 more base pairs in length. In some embodiments, the siRNA molecules are more than 70 base pairs in length. In some embodiments, the siRNA molecules are 8 to 40 base pairs in length, 10 to 20 base pairs in length, 10 to 30 base pairs in length, 15 to 20 base pairs in length, 19 to 23 base pairs in length, 21 to 24 base pairs in length. In some embodiments, the sense and antisense strands of the siRNA molecules are each independently about 19 to about 24 nucleotides in length. In some embodiments, the sense strand of an siRNA molecule is 23 nucleotides in length and the antisense strand is 21 nucleotides in length. In some embodiments, both the sense strand and the antisense strand of an siRNA molecule are 21 nucleotides in length.
After selection of a suitable target RNA sequence, siRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence, i.e., an antisense sequence, may be designed and prepared using suitable methods (see, e.g., U.S. Patent Publication Nos. 2004/0077574 and 2008/0081791 and PCT Publication No. WO 2004/016735). In some embodiments, the siRNA molecule may be single-stranded (i.e., a ssRNA molecule comprising just an antisense strand) or double stranded (i.e., a dsRNA molecule comprising an antisense strand and a complementary sense strand that hybridizes to form the dsRNA). In various embodiments, the siRNA molecules may comprise a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, comprising self-complementary sense and/or antisense strands.
In various embodiments, the antisense strand of the siRNA molecule is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In various embodiment, the antisense strand of the siRNA molecule is about 35 to about 70 nucleotides in length. In various embodiment, the antisense strand of the siRNA molecule is more than 70 nucleotides in length. In some embodiments, the antisense strand is 8 to 40 nucleotides in length, 10 to 20 nucleotides in length, 10 to 30 nucleotides in length, 15 to 20 nucleotides in length, 19 to 23 nucleotides in length, or 21 to 24 nucleotides in length.
In some embodiments, the sense strand of the siRNA molecule is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 more nucleotides in length. In various embodiments, the sense strand of the siRNA molecule is about 30 to about 70 nucleotides in length. In various embodiments, the sense strand of the siRNA molecule more than 70 nucleotides in length. In some embodiments, the sense strand is 8 to 40 nucleotides in length, 10 to 20 nucleotides in length, 10 to 30 nucleotides in length, 15 to 20 nucleotides in length, 19 to 23 nucleotides in length, 21 to 24 nucleotides in length.
In various embodiments, siRNA molecules can comprise an antisense strand comprising a region of complementarity to a target region in a target mRNA. In some embodiments, the region of complementarity is 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%, at least 99% or 100% complementary to a target region in a target mRNA. In some embodiments, the target region may comprise a region of consecutive nucleotides in the target mRNA. In some embodiments, it may not be requisite for a region of complementarity to be 100% complementary to that of its target to be specifically hybridizable or specific for a target RNA sequence.
In some embodiments, siRNA molecules disclosed herein may comprise an antisense strand that comprises a region of complementarity to a target RNA sequence and the region of complementarity is in the range of 8 to 20, 8 to 35, 8 to 45, or 10 to 50, or 5 to 55, or 5 to 40 nucleotides in length. In some embodiments, a region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary with at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, or more consecutive nucleotides of a target RNA sequence. In some embodiments, siRNA molecules comprise an antisense strand having a nucleotide sequence that contains no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches compared to the portion of the consecutive nucleotides of target RNA sequence. In some embodiments, siRNA molecules comprise a nucleotide sequence that has up to 3 mismatches over 15 bases, or up to 4 mismatches over 10 bases with a target sequence. In some embodiments, siRNA molecules comprise an antisense strand having a nucleotide sequence that has up 0, 1, 2, or 3 mismatches over 15-22 bases with a target sequence. In some embodiments, siRNA molecules comprise an antisense strand having a nucleotide sequence that has 0, 1, or 2 mismatches over 15-22 bases with a target sequence. In some embodiments, siRNA molecules comprise an antisense strand having a nucleotide sequence that has 0 or 1 mismatch over 15-22 bases with a target sequence. In some embodiments, siRNA molecules comprise an antisense strand having a nucleotide sequence that has 0 mismatches over 15-22 bases with a target sequence.
In various embodiments, siRNA molecules may comprise an antisense strand comprising a nucleotide sequence that is at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or 100% complementary to the target RNA sequence of the antisense oligonucleotides disclosed herein. In some embodiments, siRNA molecules comprise an antisense strand comprising a nucleotide sequence that is at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or 100% identical to any of the antisense oligonucleotides provided herein. In some embodiments, siRNA molecules comprise an antisense strand comprising at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, or more consecutive nucleotides of any of the antisense oligonucleotides provided herein.
In some embodiments, double-stranded siRNA can comprise sense and anti-sense RNA strands that are different lengths or the same length. In some embodiments, double-stranded siRNA molecules may also be generated from a single oligonucleotide in a stem-loop structure. The self-complementary sense and antisense regions of the siRNA molecule having a stem-loop structure may be linked by means of a nucleic acid based or a non-nucleic acid-based linker. In some embodiments, an siRNA having a stem-loop structure comprises a circular single-stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands. In some embodiments, the circular RNA may be processed in vivo or in vitro to produce an active siRNA molecule which may be capable of mediating RNAi. Small hairpin RNA (shRNA) molecules are therefore also contemplated in the present disclosure. Such molecules may comprise a specific antisense sequence together with the reverse complement (sense) sequence, which may be separated by a spacer or loop sequence in some instances. A reverse complement described herein may comprise a sequence that is a complement sequence of a reference sequence, wherein the complement sequence is written in the reverse orientation. Due to codon usage redundancy, a reverse complement can diverge from a reference sequence that encodes the same polypeptide. As used herein, “reverse complement” also includes sequences that are, e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the reverse complement sequence of a reference sequence. Cleavage of the spacer or loop can provide a single-stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule. In various embodiments, additional optional processing steps may result in removal or addition of 1, 2, 3, 4, 5 or more nucleotides from the 3′ end and/or the 5′ end of one or both strands. A spacer may be of a suitable length to allow the antisense and sense sequences to anneal and form a double-stranded structure or stem prior to cleavage of the spacer. In certain embodiments subsequent optional processing steps may result in removal or addition of 1, 2, 3, 4, 5 or more nucleotides from the 3′ end and/or the 5′ end of one or both strands. In some embodiments, a spacer sequence can be an unrelated nucleotide sequence that may be, e.g., situated between two complementary nucleotide sequence regions that, when annealed into a double-stranded nucleic acid, can comprise a shRNA.
The length of the siRNA molecules can vary from about 10 to about 120 nucleotides depending on the type of siRNA molecule being designed. Generally, between about 10 and about 55 of these nucleotides may be complementary to the RNA target sequence. For instance, when the siRNA is a double-stranded siRNA or single-stranded siRNA, the length can vary from about 10 to about 55 nucleotides, whereas when the siRNA is a shRNA or circular molecule, the length can vary from about 30 nucleotides to about 110 nucleotides.
In various embodiments, an siRNA molecule can comprise a 3′ overhang at one end of the molecule. In some embodiments, the other end can be blunt-ended or may also comprise an overhang (e.g., 5′ and/or 3′). When the siRNA molecule comprises an overhang at both ends of the molecule, the length of the overhangs may be different or the same. In some embodiments, an siRNA molecule described herein may comprises 3′ overhangs of about 1 to about 3 nucleotides on both ends of the molecule. In some embodiments, the siRNA molecule comprises 3′ overhangs of about 1 to about 3 nucleotides on both the sense strand and the antisense strand. In some embodiments, the siRNA molecule comprises 3′ overhangs of about 1 to about 3 nucleotides on the antisense strand. In some embodiments, the siRNA molecule may comprise 3′ overhangs of about 1 to about 3 nucleotides on the sense strand.
In various embodiments, the siRNA molecule comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more). In some embodiments, all of the nucleotides of the sense strand and/or the antisense strand of the siRNA molecule are modified. In certain embodiments, the siRNA molecule can comprise one or more modified nucleotides and/or one or more modified internucleotide linkages. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ end of the siRNA molecule sense strand. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ and 3′ ends of the siRNA molecule antisense strand. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ end of the siRNA molecule sense strand and at the first and second internucleoside linkages at the 5′ and 3′ ends of the siRNA molecule antisense strand.
In some embodiments, the modified nucleotide may comprise a modified sugar moiety (e.g., a 2′ modified nucleotide). In some embodiments, the siRNA molecule can comprise one or more 2′ modified nucleotides, e.g., a 2′-deoxy, 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me), 2′-O-methoxyethyl (2′-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O-N-methylacetamido (2′-O-NMA). In various embodiments, each nucleotide of the siRNA molecule can a modified nucleotide (e.g., a 2′-modified nucleotide). In some embodiments, the siRNA molecule may comprise one or more phosphorodiamidate morpholinos. In some embodiments, each nucleotide of the siRNA molecule consists of a phosphorodiamidate morpholino.
In various embodiments, the siRNA molecule may comprise a phosphorothioate or other modified internucleotide linkage. In various embodiments, the siRNA molecule may comprise, e.g., a phosphorothioate internucleoside linkage(s). In some embodiments, the siRNA molecule may comprise a phosphorothioate internucleoside linkage(s) between two or more nucleotides. In some embodiments, the siRNA molecule may comprise a phosphorothioate internucleoside linkage(s) between all nucleotides. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first, second, and/or third internucleoside linkage at the 5′ or 3′ end of the siRNA molecule. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ and/or 3′ end of the siRNA molecule. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ end of the siRNA molecule sense strand. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ and 3′ ends of the siRNA molecule antisense strand. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ end of the siRNA molecule sense strand and at the first and second internucleoside linkages at the 5′ and 3′ ends of the siRNA molecule antisense strand. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first internucleoside linkage at the 5′ and 3′ ends of the siRNA molecule sense strand, at the first, second, and third internucleoside linkages at the 5′ end of the siRNA molecule antisense strand, and at the first internucleoside linkage at the 3′ end of the siRNA molecule antisense strand.
In some embodiments, the inhibitor is a short hairpin RNA (shRNA). A “small hairpin RNA” or “short hairpin RNA” or “shRNA” described herein may include a short RNA sequence that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNAs provided herein may be chemically synthesized or transcribed from a transcriptional cassette in a DNA plasmid. The shRNA hairpin structure may be cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).
Non-limiting examples of shRNAs include a double-stranded polynucleotide molecule assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; and a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions. In some embodiments, the sense and antisense strands of the shRNA are linked by a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.
In some embodiments, the inhibitor of RNF213 protein expression comprises a site-specific nuclease. In some embodiments, the site-specific nuclease comprises a DNA nuclease such as an engineered (e.g., programmable or targetable) DNA nuclease to induce genome editing of a target DNA sequence of RNF213. Any suitable DNA nuclease can be used including, but not limited to, CRISPR-associated protein (Cas) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, other endo- or exo-nucleases, variants thereof, fragments thereof, and combinations thereof.
In some embodiments, the inhibitor of RNF213 protein expression is an antisense oligonucleotide (ASO). An ASO can downregulate a target, for example, by steric hindrance of ribosomal activity, by causing RNase H endonuclease cleavage of a target RNA, or by altering splicing or by inhibiting 5′ cap formation. An ASO can generally comprise a short nucleotide sequence which is substantially complementary to a target nucleotide sequence in a pre-mRNA molecule, an mRNA molecule, or a heterogeneous nuclear RNA (hnRNA). The degree of complementarity (or substantial complementarity) of the antisense sequence can be such that a molecule comprising the antisense sequence may form a stable double-stranded hybrid with the target nucleotide sequence in the RNA molecule. Without wishing to be bound by theory, “complementarity” of nucleic acids can mean that a nucleotide sequence in one strand of nucleic acid, e.g., due to orientation of its nucleobase groups, forms hydrogen bonds with another sequence on an opposing nucleic acid strand. The complementary bases in DNA are generally A paired with T and C paired with G. In RNA, the complementary bases are generally C with paired with G and U paired with A. Complementarity can be perfect or substantial/sufficient. Perfect complementarity between two nucleic acids means that the two nucleic acids can form a duplex in which every base within the duplex is bonded to a complementary base by Watson-Crick pairing. “Substantial” or “sufficient” complementarity means that a sequence in one strand is not completely and/or perfectly complementary to a sequence in an opposing strand but that sufficient bonding takes place between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., temperature and/or salt concentration). Such conditions can be determined by, e.g., empirical determination of Tm (melting temperature) by employing routine methods in the art or by using the sequences and standard mathematical calculations to predict the Tm of hybridized strands. Tm can include the temperature at which a population of hybridization complexes formed between two nucleic acid strands are 50% denatured (i.e., a population of double-stranded nucleic acid molecules becomes half dissociated into single strands). At a temperature below the Tm, formation of a hybridization complex can be favored, while at a temperature above the Tm, melting or separation of the strands in the hybridization complex can be favored.
In some embodiments, an ASO is a morpholino or a gapmer.
Antisense oligonucleotides can be synthetic and chemically modified.
In some embodiments, antisense oligonucleotides may be 100% complementary to the target sequence, or may comprise mismatches. so long as a heteroduplex formed between the oligonucleotide and the target sequence is sufficiently stable to tolerate the action of modes of degradation which may occur in vivo, e.g., by way of cellular nucleases. Mismatches, if present, are generally less destabilizing toward the end regions of the hybrid duplex than in the middle. The number of mismatches permitted can depend on, e.g., the percentage of G:C base pairs in the duplex, the length of the oligonucleotide, and/or the position of the mismatch(es) in the duplex, according to principles of duplex stability within the knowledge of one skilled in the art. In some embodiments, an oligonucleotide may have about 70% to about 100% sequence complementarity, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligonucleotide and the target sequence.
In some embodiments, degrading RNF213 protein comprises administering to the subject an effective amount of a proteolysis targeting chimera (PROTAC) or a molecular glue.
Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that combine a ligand for an E3 ligase with a second ligand that targets a protein of interest and catalyze its polyubiquitination and eventual proteasomal degradation. Both ends of the PROTACs are connected via a linker. First generation PROTACs used peptides to recruit a protein of interest to E3 ligases, but subsequent ones have relied on smaller and more cell-permeable synthetic ligands. These include hydroxyproline derivatives and molecules derived from thalidomide, which bind the von Hippel-Lindau protein (VHL) and cereblon (CRBN), respectively. VHL and CRBN are the substrate receptors of two cullin-RING ubiquitin ligase (CRL) complexes, namely CRL2VHL and CRL4CRBN.
Molecular glues are small molecule protein degraders that are capable of inducing interactions between an E3 ubiquitin ligase and a protein, thereby resulting in ubiquitination and degradation of the protein. Without wishing to be bound by theory, upon binding to a protein, a molecular glue can induce a conformational change and render the small molecule-protein complex a “neo-substrate” for E3 ligase. Following the formation of a ternary complex, the “neo-substrate” is ubiquitinated, which leads to ubiquitin-proteasome system (UPS)-mediated protein degradation. Non-limiting examples of molecular glues are anti-cancer aryl-sulfonamides, e.g., Indisulam and CR-83 (a CDK inhibitor). In some embodiments, a molecular glue can drive protein degradation by making novel interactions between ubiquitin ligases and neo-substrates.
The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.
HER2+ breast cancer cells with PTPN1 knockdown or treated with a PTP1B inhibitor are hypersensitive to hypoxia22. However, by studying an expanded panel of breast cancer cell lines, hypoxia hypersensitivity is not limited to the HER2+ subtype; treatment with the allosteric PTP1B inhibitor claramine also caused hypoxia hypersensitivity in several ER+ and triple negative breast cancer (TNBC) lines (
RNF213 is basally tyrosine phosphorylated in HER2+ breast cancer cells and interacts with PTP1B. “Substrate-trapping” mutants of PTP1B show increased RNF213 binding, and this interaction is competed by the active site PTP inhibitor sodium orthovanadate, suggesting that RNF213 is a PTP1B substrate22. However, RNF213 tyrosine phosphorylation is not increased in PTP1B-inhibited/deficient HER2+ breast cancer cells, indicating that only select sites are PTP1B targets. An RNF213-TurboID fusion protein was generated with the goal of identifying RNF213 binding partners by proximity ligation35, labelled interacting proteins with biotin, enriched these proteins on streptavidin beads, performed on-bead tryptic digestion, and analyzed the peptide mixture by mass spectrometry (MS). This analysis also revealed evidence of RNF213 phosphorylation on residue Y1275 (
CRISPR/Cas9 was used to generate RNF213-knockout (KO) BT474 cells and retroviral gene transduction to introduce PTPN1 or non-targeting (Control) shRNAs. This verified that phosphorylation was relevant for RNF213/PTP1B interaction. The cells were transduced with a lentivirus expressing hemagglutinin (HA) tagged-mouse Ptpn1D/A, which encodes the substrate-trapping mutant PTP1BD181A (HA-PTP1BD/A). The resultant lines were transfected with expression vectors for 3×-FLAG-RNF213 or 3×-FLAG-RNF213Y1275F. Immunoprecipitation (IP) of HA-PTP1BD/A resulted in recovery of 3×-FLAG-RNF213, while recovery of 3×-FLAG-RNF213Y1275F was reduced markedly (
PTP1B deficiency/inhibition markedly increases overall cellular ubiquitylation, dependent on RNF21322. Yet Y1275 is distant from the RING or RZ domains in the monomeric RNF213 structure26, arguing against direct regulation. The hypothesis that phosphorylation affects RNF213 oligomerization was tested by co-transfecting expression vectors for 3×-FLAG-RNF213 or 3×-FLAG-RNF213Y1275F and GFP-RNF213 and performing anti-FLAG immunoprecipitations. Notably, recovery of GFP-RNF213 in 3×-FLAG-RNF213Y1275F immunoprecipitates was impaired in BT474 (
The kinase(s) that phosphorylate(s) RNF213-Y1275 were investigated. ABL family kinase inhibitors (dasatinib, asciminib) abrogated the hypoxia hypersensitivity of BT474 PTPN1-KO cells and SKBR3 PTPN1-KO cells (
RNF213-interacting proteins were identified to evaluate how RNF213 regulates hypoxia sensitivity (
Given their role in NF-κB response, inflammation, and innate immunity, the CYLD/SPATA2 interaction with RNF213 was evaluated. Immunoblotting confirmed the interaction of C-Turbo, but not Turbo alone, with CYLD, SPATA2, and PTP1B; neither Turbo protein interacted with ERK2, which served as a control for non-specific biotinylation (
ALFA-tagged SPATA2 or CYLD and HA-Ubiquitin (HA-UB) in parental or RNF213-KO BT474 cells were co expressed to test whether CYLD and SPATA2 are RNF213 substrates. The cells were treated with claramine (10 μM) or vehicle (
3×FLAG-tagged RNF213 and various RNF213 mutants were purified from HeLa FlpIn-TRex RNF213-KO cells (
It was reported that RING-dead RNF213 or highly penetrant MMD SNPs decrease global ubiquitylation in HeLa or 293T cells and appear to act as dominant negative alleles. By contrast, the disclosure provided herein suggests that RING-dead RNF123 is an RZ gain-of-function mutation, which might be expected to increase global ubiquitylation. In an attempt to resolve this apparent discrepancy, 3×FLAG-RNF213 or RNF213 mutants were co-expressed with HA-Ubiquitin (HA-UB) in HeLa Flp-In-TRex RNF213-KO cells (
BT474 RNF213-KO (or AU565-RNF213-KO) cells were reconstituted with WT RNF213, RNF213Y1275F, RNF213K2775A (second AAA-ATPase domain mutant), RNF213H4014A (RING-dead), RNF213H4509A (RZ-dead), RNF213H4014A/H4509A (RING/RZ-dead) or RNF213K2775A/H4014A (ATPase/RZ-dead) to determine which RNF213 domains are required for hypoxia hypersensitivity. CRISPR/Cas9 was used to generate PTPN1-KO pools of each cell line. The resultant cell lines/pools were subjected to hypoxia, and lysates were analyzed by immunoblotting. WT RNF213, but not RNF213Y1275F promoted CYLD and, to a lesser extent, SPATA2 degradation in BT474 (
The effect of each mutant on hypoxia sensitivity in the presence or absence of PTPN1-KO was tested. Consistent with the above biochemical results, cells reconstituted with tyrosine phosphorylation site-, ATPase-, RZ-, compound ATPase/RZ-, or compound RING/RZ-, mutants failed to show hypoxia hypersensitivity in response to PTP1B deficiency (
Recently, the RZ domain was found to ubiquitylate Salmonella lipopolysaccharide, most likely on its lipid component29. The LUBAC complex then catalyzes further ubiquitylation, promoting bacterial clearance via autophagy. Knockdown of the LUBAC complex component RBCK1 prevented WT-RNF213-catalyzed degradation of CYLD and SPATA2 even in PTP1B-depleted BT474 or AU565 cells (
CYLD and SPATA2 are important negative regulators of NF-κB, so CYLD/SPATA2 degradation would be expected to increase NF-κB activity. A dual luciferase reporter assay was used to monitor NF-κB activity in RNF213-KO BT474 and AU565 cells reconstituted with WT RNF213 and various mutants with or without concomitant pooled PTPN1 deletion. RNF213-KO cells (Vector) failed to increase reporter activity when PTP1B was depleted, while reconstituting these cells with WT-RNF213 increased reporter activity in PTPN1-KO cells to levels similar to that of parental BT474 cells with PTPN1 deletion. RING-dead RNF213-reconstituted cells had markedly increased NF-κB activity even when PTPN1 was intact, and activity increased further upon PTPN1 deletion. Consistent with their effects on CYLD/SPATA2, RZ mutant, RING/RZ mutant, or ATPase/RZ mutant forms of RNF213 failed to induce NF-κB under any condition and might even have a dominant negative effect (
CYLD promotes apoptosis and necroptosis50,51, and NF-κB typically promotes cancer cell survival52. Yet PTP1B depletion or inhibition increases cell death in hypoxia, while promoting CYLD degradation and enhancing NF-κB activity. The mechanism that PTP1B-deficient, hypoxic cells die, was evaluated by testing the effects of known inhibitors of various cell death pathways. Neither ferroptosis nor necroptosis inhibitors blocked hypoxia hypersensitivity in PTPN1-KO cells; however, the general caspase inhibitor Z-VAD-FMK prevented hypoxia-induced cell death (
No increase in apoptosis (a caspase-dependent process) was previously observed in PTP1B-deficient breast cancer cells; instead, death appeared to be necrotic22. However, Z-VAD-FMK also inhibits “inflammatory caspases” (Caspases 1,4,5 in humans, Caspases 1 and 11 in mouse). Remarkably, treatment with VX765 (1 μM), a specific inhibitor of Caspase 1 and Caspase 4, completely blocked hypoxic cell death in PTPN1-KO BT474 cells (
NF-κB induces the expression of the inflammasome component NLRP3, as well as IL1B. The inflammasome, in turn, facilitates pro-IL-1β cleavage and release of the active cytokine, as well as the cleavage of Gasdermins53. Cleaved Gasdermins oligomerize and form pores in cell membranes, leading to pyroptotic cell death. Notably, hypoxia hypersensitivity of PTPN1-KO BT474 or MDA-MB-361 cells was also blocked by treatment with the NLRP3 inhibitor MCC950 (
NF-κB can prime cells for pyroptosis, but a “second signal” is required to trigger inflammasome assembly/inflammatory caspase activation, Gasdermin cleavage, and cell death. Several stimuli can provide the second signal, including ER stress, which is evoked by O2 levels ≤1%54. Remarkably, PTPN1-KO cells treated with the IRE1α inhibitor 4m8c were resistant to hypoxic cell death compared with DMSO control-treated cells (
Finally, to explore the pathophysiological relevance of the above-described observations, parental, PTPN1-KO, CYLD-KO, and PTPN1-KO-CYLD-KO BT474 and MDA-MB-361 cells were implanted into athymic nu nu mice and tumor development was monitored. Absence of PTP1B, CYLD, or both, markedly inhibited tumor growth (
To determine which RNF213 domains are required for tumor death, RNF213-KO sgPTPN1 BT474 cells reconstituted with doxycycline-inducible WT RNF213, RNF213Y275F, RNF213H4014A, or RNF213H4509A were implanted. Tumors were allowed to establish, but when volumes reached ˜100 mm3, mice were switched to doxycycline (Dox)-containing chow to induce WT or mutant RNF213 expression. Expression of WT or RING-dead RNF213 impaired tumor growth; by contrast, tumors expressing phosphosite- or RZ-dead mutant-reconstituted cells grew like RNF213-KO tumors (
Described herein includes unique mechanisms of inflammatory cell death in breast cancer cells exposed to hypoxia (
Hypoxic regions in tumors typically are therapy-resistant and provide a cellular reservoir for tumor recurrence3,7. PTP1B inhibition or degradation (e.g., using PROTAC technology55) can kill these hypoxic cells and could be useful in combination with other anti-neoplastics. NF-κB signaling is activated in hypoxia through IKKβ, but the detailed mechanism has remained unclear. Scholz et al.56 reported that components of the TRAF6 complex (OTUB, UVEV1a) associate with PHD1 and that multiple NF-κB pathway components are ubiquitylated. The results described herein indicate that hypoxia also promotes NF-κB activation via degradation of a major deubiquitylase complex. HPV-infected cells activate NF-κB in hypoxia via CYLD degradation57. Degradation requires E6, but not E6AP, the ubiquitin ligase that typically mediates E6 action.
Importantly, PTP1B inhibition/deficiency induces pyroptosis, an inflammatory form of cell death, which could help “turn cold tumors hot” and promote the anti-tumor immune response53. Inflammation also can suppress tumor immunity53; notably, PTP1B inhibition/deficiency increases IL-1β production, which promotes immunosuppression by myeloid cells58. PTP1B also has direct effects in myeloid cells59 and lymphocytes60,61; whether the PTP1B/RNF213/CYLD/SPATA2 pathway is active in these cells remains unclear. A PTP1B/TCPTP inhibitor is in clinical trials as an anti-neoplastic agent34 (NCT04777994), mainly because of its predicted immunostimulatory effects60,62. Elucidating the cell-autonomous and non-autonomous effects of such agents, including if/how TCPTP inhibition affects RNF213, should aid in their rational deployment. It also will be important to test whether PTP1B controls hypoxia sensitivity in other tumor types, particularly pancreas cancer, which is notoriously hypoxic63.
Most previous studies of RNF213 have focused on its E3-ubiquitin ligase domains, although it is known that its E3 ligase activity requires ATPase function and, most likely, hexamerization29. Oligomerized RNF213 serves as a sensor for ISG1564; however, reciprocal control of Y1275 phosphorylation by ABL1/2 and PTP1B is the first example of a signaling pathway regulating RNF213 oligomerization/E3 activation. The RZ domain, previously implicated in lipid ubiquitylation, was found to have protein targets (CYLD/SPATA2).
The results described herein also yield several new clues into MMD pathogenesis. RNF213-catalyzed CYLD/SPATA2 degradation provides a mechanistic explanation for earlier reports that RNF213, and to a greater extent, MMD-associated SNPs, activate NF-κB30. MMD is characterized by precocious carotid occlusions and stroke. The most common RNF213 SNPs activate the RZ domain and confer LUBAC-independence on CYLD/SPATA2 degradation (and consequently, NF-κB activation and increased inflammation) could explain how these alleles promote vascular disease. MMD is also associated with Graves' disease, systemic lupus erythematosus, Sjögren's syndrome, multiple sclerosis, rheumatoid arthritis, and other immune/inflammatory disorders65,66. Bypassing the LUBAC complex requirement for CYLD/SPATA2 degradation/NF-κB signaling could help explain these associations.
Finally, disordered regulation of SPATA2/CYLD and possibly other RNF213 interactors could be relevant to more common diseases and might be targeted therapeutically. For example, RNF213 deficiency abrogates lipotoxicity caused by saturated fatty acids in vitro32. Saturated fatty acids also can provide the “second signal” for pyroptosis67 and induce ER stress68. In some embodiments, RNF213 degraders, ATPase inhibitors, or RZ inhibitors might have therapeutic benefit in, for example, non-alcoholic fatty acid disease. Several other RNF213 interactors (MAP4K4, SPTLC1, SARS2) are associated with atherosclerotic disorders including stroke.
As shown in
Previously, it has been reported that RNF213 increases global ubiquitylation in absence or inhibition of PTP1B. Since TNK1 is a major tyrosine kinase that could affect global ubiquitylation as it directly binds to ubiquitin, whether TNK1 regulates global ubiquitination in PTP1B inhibited cells was tested. The results showed that TNK1-KD suppressed the increased global ubiquitylation in PTP1B inhibited cells (
Since RNF213 promotes hypoxia hypersensitivity in PTPN1-KO cells, the effects of TNK1-KD in PTPN1-KO cells on hypoxia hypersensitivity was tested. The results suggested that TNK1-KD impaired the hypoxia hypersensitivity of BT474 PTPN1-KO cells (top) and MDA-MB-361 PTP1B-KO cells (bottom) (
Further, whether the effects of TNK1 on hypoxia viability were regulated through RNF213 phosphorylation was tested. For this, purified RNF213 was subjected to an in vitro kinase assay. The results suggested that TNK1 phosphorylates RNF213 in a dose dependent manner (
Overall, results described in the present Example suggest that TNK1 phosphorylates RNF213 and positively regulates RNF213 activity.
Below are the methods used in the Examples described above.
Sources of the breast cancer cell lines used here were described69; all are maintained as frozen stocks by the Neel Laboratory. Except where indicated, cells were cultured in DMEM+10% FBS with 100 U/ml penicillin and 100 mg/ml streptomycin at 37° C. in 5% CO2 and tested monthly for mycoplasma by PCR.
Isogenic HeLa Flp-In T-Rex RNF213-KO cells that inducibly express wild-type RNF213 or RNF213 variants in response to doxycycline (DOX) were generated by co-transfecting pcDNA5/FRT/TO-based expression constructs with 3×-Flag tagged RNF213 or RNF213 variant with pOG44, which directs constitutive expression of Flp recombinase (FLP). were selected by adding hygromycin B (200 μg/ml) 48 hours post-transfection. Single cell clones of RNF213-deleted HeLa, BT474, and AU565 cells, or of PTPN1-deleted BT474, SKBR3, and MDAMB361 cells, were generated by using CRISPR/Cas9 technology70. Briefly, sgRNAs targeting each gene were cloned into the BbsI site of pSpCas9 (BB)-2A-Puro (PX459; Addgene), and cells were transfected with each clone using Lipofectamine™ 3000 Transfection Reagent (ThermoFisher Scientific). After 48 hours, cells were diluted into 96-well plates (1 cell/well) containing 48-hour conditioned media from the same line in addition to standard media (1:1). Knockout clones were confirmed by immunoblotting. The sgRNA sequences are shown in Table 2.
Piggbac (PB)-based transposon expression was used to re-express WT RNF213 or RNF213 variants under Dox control in RNF213-KO BT474 and RNF213-KO AU565 cells. Briefly, PB-TA-ERN (Addgene)-based expression constructs containing RNF213 and RNF213 variants were co-transfected with transposase expression vector, and integrants were selected by adding G418 (800 μg/ml). RNF213 expression was induced by adding 0.5 gig/ml Dox.
PTPN1 depletion was accomplished by stable shRNA knockdown using pSUPER-Puro-based retroviral vectors. RBCK1, ABL1, and ABL2 siRNA (Horizon Discovery) were introduced using Lipofectamine™ RNAiMAX reagent (ThermoFisher Scientific). Pooled knockout lines were generated by infection with lentiviruses expressing CAS9 and appropriate sgRNAs. Lentiviruses and retroviruses were packaged as described71.
Cells were seeded in 6-well plates (2×106/well for MDAMB361, 1.5×106/well for other lines) and maintained in at 37° C., 500 CO2. After 24 hours, viable cell number was determined by Trypan Blue exclusion, and fresh media was added to replicates placed in a standard incubator (normoxia) and in hypoxic conditions (0.1% O2, Whitley H35 Hypoxystation). Viable cells were counted at various times, as indicated.
RNF213 mutants were generated by PCR or by using fragments synthesized by Genewiz/IDT. Fragments containing the desired mutation were sequenced, digested, and cloned into wild-type RNF213 plasmids by using T4 DNA ligase or via Gibson assembly72.
Cells co-transfected with an NF-κB reporter construct (p2×-NF-κB-BS-Luc-Firefly, a generous gift of Dan Littman, NYU Grossman School of Medicine) and pCMV-Renilla, were subjected to 0.1% O2 for the indicated times. Firefly and Renilla luciferase was measured using the Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer's instructions. Reporter activity was quantified as the ratio of Firefly/Renilla signal, and all signals were normalized to the signal in parental BT474 cells.
Cell supernatants were collected at the indicated times, and IL-1 levels were quantified by using the Lumit™ IL-10 Immunoassay (Promega), according to manufacturer's instructions.
Cells were lysed in RIPA (total cell lysates, ALFA-tag precipitations) or NP40 buffer (co-immunoprecipitations); buffer compositions and immunoprecipitation conditions are described below27. ALFA-tagged proteins were recovered using a single domain camelid antibody bound to magnetic beads (NanoTag Biotechnologies). Antibody details and concentrations used are summarized in Table 3.
Cells were cultured in standard media but with 10% tetracycline (Tet)-free FBS (Clonetech) for at least 1 week. Tet-free/biotin-free FBS was generated by incubating Tet-free FBS with sterile streptavidin beads overnight followed by centrifugation to remove the beads. N-Turbo, C-Turbo, and Turbo proteins were induced by adding 1 μg/ml Dox to cells for 36 hours. For the last ten min. of incubation, 500 μM of biotin was added to the medium. Labelling reactions was terminated by removing the medium and adding ice-cold PBS to plates, which were kept on ice. Lysates were collected in RIPA buffer without Na-deoxycholate, and biotin-labelled proteins were collected by incubating with high-capacity streptavidin agarose beads for 4 hours at 4° C. Comparable protein amounts (assessed by silver stain and avidin blotting) were analyzed by MS.
HeLa RNF213-KO cells expressing RNF213 or RNF213 variants (except RNF213H4509A) were induced with 0.5 μg/ml Dox for 4 days. The 3×-FLAG-RNF213H4509A variant was transiently transfected into HeLa RNF213-KO cells, as the stable expression of this variant was low. Lysates from ten 15 cm plates were subjected to anti-FLAG immunoprecipitation. RNF213 species were eluted with 3×-FLAG peptide and concentrated by passage through an Amicon Ultra-4 Centrifugal Filter (100 kDa cut-off).
Ubiquitylation assays were performed as described27. Briefly, 0.25 μM of purified RNF213 or RNF213 variant were incubated with 2.5 μM GST-SPATA2 (Abnova) or 2.5 μM 6×His-CYLD (R&D) in E3-Ligase buffer (R&D) along with 50 μM HA-Ub, 100 nM UBE1 (E1), 1 μM UBE2L3 (E2), and 10 mM Mg-ATP (all from Boston Biochem) for 30 min. in a total reaction volume of 25 μl. Reactions were terminated by adding SDS-PAGE sample buffer followed by incubation for 5 min. at 95° C.
Purified full-length 3×-FLAG-tagged RNF213 or RNF213Y1275F were mixed with varying amounts of bacterially produced His-ABL1 (as indicated) and incubated with 600 ng of RNF213 in kinase assay buffer (50 mM HEPES [pH 7.5], 10 mM MgCl2, 2.5 mM DTT, 75 μM ATP) for varying times, as indicated. Final reaction volumes were 20 μl. Reactions were terminated by adding SDS-PAGE sample buffer followed by incubation for 5 min. at 95° C.
Proteins collected on streptavidin beads were digested with sequencing grade trypsin and analyzed by LC/MS/MS using a Thermo Scientific EASY-nLC 1200 and a Thermo Scientific Orbitrap Eclipse Mass Spectrometer MS/MS spectra were searched against the Uniprot human database with the addition of the sequences of RNF213 with TurboID at the N- and C-terminus using Sequest within Proteome Discoverer 1.4. MS/MS spectra were also searched using Byonic by Protein Metrics specifically to identify phosphorylation sites on RNF213.
Graphs were generated and statistical analyses were performed using GraphPad Prism9. Multiple groups were compared by two-way ANOVA, with additional Tukey multiple comparisons test. Two group comparisons were evaluated by two-tailed t test. Values represent the mean of experiments, with error bars representing S.E.M.
For co-immunoprecipitations, cells were lysed in NP40 buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 2 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 1% [vol/vol] NP-40, 40 μg/ml phenylmethyl sulfonyl fluoride, 2 μg/ml antipain, 2 μg/ml pepstatin A, 20 μg/ml leupeptin, and 20 μg/ml aprotinin). For streptavidin affinity purifications and standard immunoprecipitations, modified RIPA buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 2 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 10% [vol/vol] NP-40, 0.10% (wt/vol) SDS, 40 μg/ml phenylmethyl sulfonyl fluoride, 2 μg/ml antipain, 2 μg/ml pepstatin A, 20 μg/ml leupeptin, and 20 μg/ml aprotinin) was used for cell lysis. For detection of ubiquitylated species in vivo, 10 mM N-ethylmaleimide (NEM) and 10 mM iodoacetamide (IAM) were added to the lysis buffer.
For immunoblotting of whole cell lysates, equal amounts of protein per sample were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). For immunoprecipitation of FLAG-tagged proteins, lysates were incubated with anti-FLAG-magnetic beads for 4 hours on a rotator-mixer at 4° C. For immunoprecipitation of HA-tagged proteins, lysates were incubated with anti-HA-agarose beads for 4 hours on a rotator-mixer at 4° C. For immunoprecipitation of ALFA-tagged proteins, lysates were incubated with ALFA-selector ST agarose beads overnight on a rotor-mixer at 4° C. For recovery of biotinylated proteins, high-capacity streptavidin agarose resin (ThermoScientific) was added to lysates, which were rotated for 4 hours at 4° C. Beads were washed five times in lysis buffer, incubated in sample buffer at 95C for 5 min. and resolved by SDS-PAGE and immunoblotting. For in vitro ubiquitylation and phosphorylation using purified RNF213 and RNF213 variants, proteins were eluted from beads by incubation with an excess of 3×-FLAG peptide. Lysates and immunoprecipitates were resolved by modified SDS-PAGE on 3-8% Tris-acetate gels (Novex) or 5% Tris-glycine gels. Gels were transferred in 1× transfer buffer, 10% methanol for 1 hour at 25 V using an XCell II Blot Module (Novex) and analyzed by acquisition software (Image Studio Lite), on an Odyssey Infrared imaging system (Li-Cor Biosciences). All antibodies used are listed in Table 3 above.
Proteins bound to streptavidin agarose beads were washed with 100 mM NH4HCO3 (pH 8), reduced with 2.5 μL of 0.2M dithiothreitol at 57C for 1 hour, alkylated with 2.5 μL of 0.5M IAM for 45 min. at room temperature in the dark, and digested overnight at room temperature with 500 ng sequencing grade modified trypsin (Promega). After digestion, samples were acidified to a final concentration of 0.5% trifluoroacetic acid (TFA). A C18 cleanup was performed on all samples using Ultra-Micro SpinColumns (Harvard Apparatus). Briefly, samples were loaded onto equilibrated spin columns, rinsed with 0.1% TFA, and eluted with 40% acetonitrile/0.5% acetic acid and 80% acetonitrile/0.5% acetic acid. Organic solvent was removed using a SpeedVac concentrator, and all samples were reconstituted in 0.5% acetic acid.
One-third of each sample was analyzed individually by LC/MS/MS. Samples were separated online using a Thermo Scientific EASY-nLC 1200, where solvent A was 2% acetonitrile/0.5% acetic acid, and solvent B was 80% acetonitrile/0.5% acetic acid. A 60-min. gradient from 5-35% B was applied to all samples. Peptides were gradient-eluted directly to a Thermo Scientific Orbitrap Eclipse Mass Spectrometer. High resolution, full MS spectra were acquired with a resolution of 240,000, an AGC target of 1e6, a maximum ion time of 50 ms, and a scan range of 400 to 1500 m/z. Following each full MS scan, data dependent HCD MS/MS spectra collected with low resolution at top speed with a 3 ms cycle time. All MS/MS spectra were collected with a rapid scan in the ion trap, an AGC target of 2e4, a maximum ion time of 18 ms, one microscan, 0.7 m/z isolation window, auto scan range mode, and NCE of 27. MS/MS spectra were searched against the Uniprot human database using Sequest within Proteome Discoverer. 1.4. MS/MS spectra were also searched with Byonic by Protein Metrics against a database containing just RNF213 and common contaminants to identify phosphorylation sites.
For TurboID analyses, beads were washed two times with 100 mM NH4HCO (pH 8). Samples were reduced with 2 μL of 0.2M dithiothreitol at 57C for 1 hour and alkylated with 2 μL of 0.5M IAM for 45 min. at room temperature in the dark. Samples were digested overnight at room temperature with 250 ng sequencing trypsin. After digestion, samples were acidified to a final concentration of 0.5% trifluoroacetic acid (TFA) and cleaned up on C18 columns as above.
One-third of each sample was analyzed individually by LC/MS/MS. LC conditions were identical to those above. High resolution full MS spectra were acquired with a resolution of 70,000, an AGC target of 1e6, a maximum ion time of 120 ms, and scan range of 400 to 1500 m/z. Following each full MS were 20 data-dependent high resolution HCD MS/MS spectra. All MS/MS spectra were collected with resolution of 17,500, an AGC target of 5e4, a maximum ion time of 120 ms, one microscan, 2 m/z isolation window, fixed first mass of 150 m/z, and NCE of 27. MS/MS spectra were searched against the Uniprot human database with the added sequences of RNF213 with the TurboID on the N- and C-terminus using Sequest within Proteome Discoverer 1.4.
Additional chemicals, kits, and reagents used in above-described methods are provided in Table 4. Examples of plasmids used in the above-described methods are provided in Table 5A. Examples of primers used in the above-described methods are provided in Table 5B.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.
Homo sapiens OX = 9606 GN = ERCC2 - [ERCC2_HUMAN]
Homo sapiens OX = 9606 GN = PDXDC1 - [PDXD1_HUMAN]
Homo sapiens OX = 9606 GN = ZGPAT - [ZGPAT_HUMAN]
Homo sapiens OX = 9606 GN = PDPR PE = 1 SV = 2 - [PDPR_HUMAN]
Homo sapiens OX = 9606 GN = EIF2AK1 - [E2AK1_HUMAN]
Homo sapiens OX = 9606 GN = PIK3C2A PE = 1 SV = 2 - [TSTD2_HUMAN]
Homo sapiens OX = 9606 GN = TGFB111 - [TGFI1_HUMAN]
Homo sapiens OX = 9606 GN = RPUSD2 - [RUSD2_HUMAN]
Homo sapiens OX = 9606 GN = CLPTM1 - [CLPT1_HUMAN]
Homo sapiens OX = 9606 GN = HMGA1 - [HMGA1_HUMAN]
Homo sapiens OX = 9606 GN = FDXR - [ADRO_HUMAN]
Homo sapiens OX = 9606 GN = CPT1A - [CPT1A_HUMAN]
Homo sapiens OX = 9606 GN = LRBA - [LRBA_HUMAN]
Homo sapiens OX = 9606 GN = STAT2 - [STAT2_HUMAN]
Homo sapiens OX = 9606 GN = IFIT5 PE = 1 SV = 1 - [IFIT5_HUMAN]
Homo sapiens OX = 9606 GN = PDE3B - [PDE3B_HUMAN]
Homo sapiens OX = 9606 GN = RNPS1 - [RNPS1_HUMAN]
Homo sapiens OX = 9606 GN = OASL - [OASL_HUMAN]
Homo sapiens OX = 9606 GN = ALDH1L2 PE = 1 SV = 2 - [AL1L2_HUMAN]
Homo sapiens OX = 9606 GN = PARP14 - [PAR14_HUMAN]
Homo sapiens OX = 9606 GN = WDR45B PE = 1 SV = 2 - [WIPI3_HUMAN]
Homo sapiens OX = 9606 GN = DNTTIP2 PE = 1 SV = 2 - [TDIF2_HUMAN]
Homo sapiens OX = 9606 GN = MFSD6 PE = 1 SV = 2 - [MFSD6_HUMAN]
Homo sapiens OX = 9606 GN = PNPT1 PE = 1 SV = 2 - [PNPT1_HUMAN]
Homo sapiens OX = 9606 GN = STRBP - [STRBP_HUMAN]
Homo sapiens OX = 9606 GN = ALG1 PE = 1 SV = 2 - [ALG1_HUMAN]
Homo sapiens OX = 9606 GN = ASPSCR1 - [ASPC1_HUMAN]
Homo sapiens OX = 9606 GN = MKRN2 - [MKRN2_HUMAN]
Homo sapiens OX = 9606 GN = AZI2 - [AZI2_HUMAN]
Homo sapiens OX = 9606 GN = EML4 PE = 1 SV = 3 - [EMAL4_HUMAN]
Homo sapiens OX = 9606 GN = PHF8 - [PHF8_HUMAN]
indicates data missing or illegible when filed
This patent application claims priority to U.S. Provisional Application No. 63/524,807, filed on Jul. 3, 2023, the disclosure of which is herein incorporated by reference in its entirety.
This invention was made with government support under P30 CA016087, R01 CA264933, and R01 CA049152 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63524807 | Jul 2023 | US |
