EP300 DEGRADERS AND THEIR USE IN NEUROBLASTOMA - Patent application

Abstract

The present invention relates to methods for treating diseases or disorders associated with EP300 dependence and elevated CRBN expression levels, such as cancer (eg, neuroblastoma).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 USC § 119(e) to U.S. Provisional Application No. 63 / 158,620, filed March 9, 2021, which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCHII format and is incorporated herein by reference in its entirety. The ASCII copy, created on March 8, 2022, is named 52095-7180001WO_ST25.txt and is 72 kilobytes in size. [Background technology]

[0003] Various studies suggest that E1A binding protein P300 (EP300, KAT3B) and cAMP responsive element binding protein (CREB)-binding protein (CBP, CREBBP, KAT3A) play overlapping but distinct roles in regulating cell survival. Germline loss of EP300 or CBP results in mouse embryonic lethality with distinct phenotypes (Yao et al. Cell 93:361-72 (1998)). Furthermore, CBP is required for self-renewal, whereas EP300 is required for differentiation of hematopoietic stem cells (Rebel et al. Proc.Natl. Acad. Sci. USA 99:14789-94 (2002)). Somatic mutations in either EP300 or CBP are found in a variety of malignancies, including neuroblastoma, and loss of EP300 in CBP-mutated tumor cells is synthetically lethal (Barretina et al. Nature 483:603-7 (2012); Ogiwara et al. Cancer Discov. 6:430-45 (2016)).

[0004] Chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq) studies have identified overlapping but distinct DNA binding sites genome-wide for EP300 and CBP, indicating that these two proteins may have distinct functions by regulating enhancers of distinct genes (Martire et al. BMC Mol. Cell Biol. 21:55 (2020); Ramos et al. Nucleic Acids Res. 38:5396-5408 (2010)). Many studies examining EP300 and CBP have relied on genetic disruption or mRNA depletion of each gene, thereby precluding time-related analysis, or have relied on the use of inhibitors with non-selective activity against both enzymes (Dancy and Cole, Chem. Rev. 115:2419-52 (2015); Hammitzsch et al. Proc.Natl. Acad. Sci. USA 112:10768-173 (2015); Lasko et al. Nature 550:128-2 (2017); Yan et al. J. Invest. Dermatol. 133:2444-52 (2013); Zucconi et al. Biochemistry 55:3727-34 (2016)). Thus, induction of pharmacological inhibitors that target only one of these enzymes is limited by the homology between these proteins (Dancy and Cole, Chem. Rev. 115:19-2452 (2015); Lasko et al. Nature 550:128-32 (2017)). Summary of the Invention [Means for solving the problem]

[0005] The present invention is based on the surprising discovery that EP300, but not its paralog CREB binding protein (CBP), is required for the regulation of key enhancers in high-risk neuroblastoma. EP300 is an enhancer-regulated dependent in neuroblastoma (NB) and is recruited to DNA via interaction with transcription factor activator protein 2B (TFAP2β). Targeted pharmacological degradation of EP300 by the proteolytic targeting chimera (PROTAC®) JQAD1 resulted in a global loss of histone acetylation in neuroblastoma cell lines. Degradation of EP300 drives neuroblastoma apoptosis, in part due to loss of MYCN chromatin localization, and limits toxicity to non-transformed cells. Functional genomic and chemical analyses have revealed widespread dependence on EP300 in many types of human cancers, including myeloma, lymphoma, leukemia, melanoma, rhabdomyosarcoma, colon, rectal, gastric, breast, brain, and pancreatic cancers.

[0006] A method for treating a subject, e.g., a human subject, having a disease or disorder associated with EP300 dependence and elevated cereblon (CRBN) expression levels is carried out by obtaining a test sample from a subject having or at risk of developing the disease; identifying an increased expression level of CRBN in the test sample compared to a reference sample; and administering a therapeutically effective amount of a selective degrader of EP300 to the subject, thereby treating the disease or disorder.

[0007] In one aspect, the disease or disorder is cancer.In certain embodiments, the cancer is solid tumor (i.e., tumor that lacks any liquid or cyst), such as neuroblastoma, rhabdomyosarcoma, malignant melanoma, colon cancer, rectal cancer, gastric cancer, breast cancer, brain cancer, and pancreatic cancer.In certain embodiments, the cancer is hematological cancer (i.e., cancer that affects blood, bone marrow, and lymph nodes), such as leukemia, myeloma, and lymphoma.In certain embodiments, the cancer is high-risk neuroblastoma.

[0008] For example, the test sample is obtained from tumor tissue or tumor microenvironment. Alternatively, the test sample is obtained from body fluid, such as plasma, blood, urine, sputum, or cerebrospinal fluid (CSF). Other exemplary body fluids include serous fluid (e.g., pleural fluid, peritoneal fluid, and pericardial fluid), synovial fluid, and drainage and dialysis fluid.

[0009] In one embodiment, the reference sample is obtained from a healthy normal tissue or a tumor tissue. For example, the reference sample is obtained from a healthy normal tissue from the same individual as the test sample or one or more healthy normal tissues from a different individual.

[0010] In some cases, whether EP300 is required for tumor growth, i.e., whether the tumor is EP300-dependent, is identified by CRISPR-Cas9-mediated knockout of EP300 in cells of the test sample.

[0011] In some cases, the expression level of CRBN is detected via Affymetrix Gene Array hybridization, next-generation sequencing, ribonucleic acid sequencing (RNA-seq), real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) assays, immunohistochemistry (IHC), or immunofluorescence.

[0012] In one embodiment, the selective degrader of EP300 is JQAD1 or a pharma- ceutically acceptable salt thereof.

[0013] Preferably, tumor cell viability, tumor cell proliferation, or tumor metastasis is inhibited, for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0014] Optionally, tumor cell proliferation is reduced, e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In another embodiment, tumor cell apoptosis is induced.

[0015] In some cases, the method further comprises administering to the subject a chemotherapeutic agent, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or stem cell transplant. For example, chemotherapeutic agents include cis-retinoic acid, cyclophosphamide (Cytoxan®, Neosar®, Endoxan®), cisplatin (Platinol®), carboplatin (Paraplatin®), vincristine (Oncovin®, Vincasar PFS®, VCR), doxorubicin (Adriamycin®, Rubex®), etoposide (Toposar®, VePesid®, Etopophos®, VP-16), topotecan (Hycamtin®), busulfan (Myleran®, Busulfex®), and melphalan (Alkeran®, L-PAM, Evomela®), or thiotepa (Thioplex®, Tepadina®).

[0016] In one embodiment, the chemotherapeutic agent is administered with a steroid, for example, the steroid is prednisone (Sterapred®, Prednisone Intensol) or dexamethasone (Degadrone®).

[0017] In some cases, the method further comprises administering a combination chemotherapy agent to the subject. For example, the combination chemotherapy agent comprises carboplatin (Paraplatin®) or cisplatin (Platinol®), cyclophosphamide (Cytoxan®, Neosar®, Endoxan®), doxorubicin (Adriamycin®, Rubex®), and etoposide (Toposar®, VePesid®, Etopophos®, VP-16), or irinotecan (Onivyde®), temozolomide (Temodal®), or ifosfamide (Ifex®). In some cases, the treatment is followed by stem cell transplantation.

[0018] In some cases, the methods further include administering to the subject an immunosuppressant, such as dinutuximab (Unituxin®), with or without cis-retinoic acid.

[0019] Also provided is a method for determining whether degradation of EP300 in a subject having cancer provides a clinical benefit in the subject, the method comprising the steps of obtaining a test sample from a subject having cancer or at risk of developing cancer, determining the expression level of CRBN in the test sample, comparing the expression level of CRBN to the expression level of CRBN in a reference sample, and determining whether EP300 degradation inhibits cancer in the subject if the expression level of CRBN in the test sample differs from the expression level of CRBN in the reference sample.

[0020] For example, the test sample is obtained from tumor tissue or tumor microenvironment. Alternatively, the test sample is obtained from body fluid (e.g., plasma, blood, urine, sputum, or CSF). Other exemplary body fluids include serous fluid (e.g., pleural fluid, peritoneal fluid, and pericardial fluid), synovial fluid, and drainage and dialysis fluid.

[0021] In one embodiment, the reference sample is obtained from healthy normal tissue.

[0022] For example, clinical benefit in a subject includes a complete or partial response as defined by response evaluation criteria in solid tumors (RECIST), stable disease as defined by RECIST, or long-term survival despite disease progression or response as defined by irRC criteria.

[0023] In one example, the test sample is obtained from cancer tissue, and the method further includes determining that degradation of EP300 in a subject having cancer confers a clinical benefit in the subject if the expression level of CRBN in the test sample is equal to or greater than the level of CRBN in the reference sample.

[0024] In another case, the test sample is obtained from cancer tissue and the method further includes determining that degradation of EP300 in a subject having cancer does not result in a clinical benefit in the subject if the expression level of CRBN in the test sample is lower than the level of CRBN in the reference sample.

[0025] definition Unless specifically stated or clear from the context, the term "about" as used herein is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless clear from the context, all numerical values ​​provided herein are modified by the term "about".

[0026] The phrase "aberrant expression" is used to refer to an expression level that deviates from a normal reference expression level of a gene (ie, an increased or decreased expression level).

[0027] By "agent" is meant any small chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

[0028] By "alteration" is meant a change (increase or decrease) in expression levels or activity of a gene or polypeptide, as detected by standard art-known methods such as those described herein. As used herein, alteration includes at least a 1% change in expression levels, e.g., at least a 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% change in expression levels. For example, alteration includes at least a 5%-10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

[0029] The term "antibody" (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0030] To "bind" to a molecule means to have a physicochemical affinity for that molecule.

[0031] "Control" or "reference" refers to a standard of comparison. In one aspect, as used herein, a sample or subject that is "altered compared to a control" is understood to have a level that is statistically different from a normal sample, an untreated sample, or a sample from a control sample. A control sample includes, for example, a cultured cell, one or more laboratory test animals, or one or more human subjects. How to select and test a control sample is within the capabilities of a person skilled in the art. The analyte can be a naturally occurring substance (e.g., an antibody, a protein) that is characteristically expressed or produced by a cell or organism, or a substance produced by a reporter construct (e.g., β-galactosidase or luciferase). Depending on the method used for detection, the amount and measurement of the change can vary. Determining statistical significance is within the capabilities of a person skilled in the art, for example, the number of standard deviations from the mean that constitute a positive result.

[0032] As used herein, the term "pharmaceutically acceptable" in the context of salt refers to a salt of a compound that does not abolish the biological activity or properties of the compound and is relatively non-toxic, i.e., the salt form of the compound can be administered to a subject without causing undesired biological effects (such as dizziness or stomach upset) or interacting in a harmful manner with any of the other components of the composition in which it is contained. The term "pharmaceutically acceptable salt" refers to the product obtained by reacting the compound of the present invention with a suitable acid or base. Examples of pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic bases (e.g., Li, Na, K, Ca, Mg, Fe, Cu, Al, Zn and Mn salts). Examples of pharma- ceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids, such as, for example, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, 4-methylbenzenesulfonate or p-toluenesulfonate, etc. Certain compounds of the present invention can form pharma- ceutically acceptable salts with various organic bases, such as lysine, arginine, guanidine, diethanolamine or metformin.

[0033] The terms "effective amount" and "therapeutically effective amount" of a formulation or formulation component refer to an amount of the formulation or component, alone or in combination, sufficient to provide a desired effect. For example, "effective amount" refers to the amount of a compound, alone or in combination, required to improve the symptoms of a disease, e.g., NB, compared to an untreated patient. The term "therapeutically effective amount" includes an amount of a compound, alone or in combination, that, when administered, is sufficient to induce a positive modification in a disease (e.g., NB) (e.g., degrade EP300 in diseased cells), or to prevent the onset or progression of a disease, or to alleviate at least to some extent one or more of the symptoms of a disease in a subject. The effective amount of the active compound(s) used to practice the present invention for the therapeutic treatment of a disease varies depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosing regimen. Such an amount is referred to as an "effective" amount.

[0034] The term "expression profile" is used broadly to include genomic expression profile. The profile can be generated by any convenient means for determining the level of nucleic acid sequences, such as quantitative hybridization of microRNA, labeled microRNA, amplified microRNA, complementary / synthetic DNA (cDNA), etc., quantitative polymerase chain reaction (PCR), and ELISA for quantification, allowing the analysis of differential gene expression between two samples. A tumor sample from a subject or patient is assayed. The sample is collected by any convenient method known in the art. According to some embodiments, the term "expression profile" refers to measuring the relative abundance of nucleic acid sequences in the measured sample.

[0035] The nucleic acid molecule useful in the method of the present invention includes any nucleic acid molecule that encodes the polypeptide of the present invention or its fragment.Such a nucleic acid molecule does not need to be 100% identical to an endogenous nucleic acid sequence, but typically shows substantial identity, for example, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity.A polynucleotide that has "substantial identity" to an endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule.

[0036] As used herein, "obtaining," as in "obtaining an agent," includes synthesizing, purchasing, or otherwise acquiring the agent.

[0037] As used herein, unless otherwise stated or clear from context, the term "or" is understood to be inclusive. As used herein, unless otherwise stated or clear from context, the terms "a," "an," and "the" are understood to be singular or plural.

[0038] The phrase "pharmaceutically acceptable carrier" is art-recognized and includes pharmaceutically acceptable substances, compositions, or vehicles suitable for administering the compounds of the present invention to mammals. Suitable carriers may include, for example, liquids (both aqueous and non-aqueous, and combinations thereof), solids, encapsulating materials, gases, and combinations thereof (e.g., semi-solids), and gases that function to carry or transport the compound from one organ or body part to another. A carrier is "acceptable" in the sense that it is physiologically inert and compatible with the other ingredients of the formulation, and not harmful to the subject or patient. Depending on the type of formulation, the composition may further include one or more pharmaceutically acceptable excipients. Examples of materials which may function as pharma- ceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and other non-toxic compatible substances used in pharmaceutical preparations.

[0039] By "protein" or "polypeptide" or "peptide" is meant any chain of more than two natural or unnatural amino acids that constitutes all or part of a naturally occurring or non-naturally occurring polypeptide or peptide, as described herein, regardless of post-translational modification (e.g., glycosylation or phosphorylation).

[0040] The terms "prevent" and "prevention" refer to the administration of an agent or composition to a clinically asymptomatic individual who is at risk of, susceptible to, or predisposed to developing a particular adverse condition, disorder, or disease, and thus relates to the prevention of the onset of symptoms and / or their underlying causes.

[0041] The terms "prognosis," "staging," and "determination of aggressiveness" are defined herein as a prediction of the degree of severity of a neoplasm, such as NB, and a prediction of its progression, as well as the chances of recovery expected from the usual course of the disease. Once aggressiveness is determined, an appropriate treatment method is selected.

[0042] Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value forms another embodiment. It is further understood that each of the endpoints of a range is significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. It is also understood that throughout this application, data is provided in several different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, when a specific data point "10" and a specific data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are also disclosed, as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0043] It is understood that the ranges provided herein are shorthand for all values ​​within the range.For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 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, 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, as well as all fractional values ​​between the aforementioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from either end of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0044] By "reduce" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0045] By "specifically binds" is meant a compound or antibody that recognizes and binds to a polypeptide of the invention, but does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample that naturally contains a polypeptide of the invention.

[0046] By "selective degrader" is meant a bifunctional compound or PROTAC® (e.g., JQAD1) that preferentially binds to and recruits a specific protein (e.g., EP330) for targeted proteasomal degradation.

[0047] A subject "suffering or suspected of suffering from" a particular disease, condition or syndrome has a sufficient number of risk factors or shows a sufficient number or combination of signs or symptoms of a disease, condition or syndrome so that a qualified individual diagnoses or suspects that the subject suffers from the disease, condition or syndrome. Methods for identifying subjects suffering from or suspected of suffering from a condition associated with EP300 dependency and elevated CRBN expression levels (e.g., cancer (e.g., NB)) are within the capabilities of those skilled in the art. Subjects suffering from and suspected of suffering from a particular disease, condition or syndrome are not necessarily two separate groups.

[0048] As used herein, "susceptible to" or "prone to" or "predisposed to" or "at risk of developing" a particular disease or condition refers to an individual who, based on genetic, environmental, health, and / or other risk factors, is more likely to develop the disease or condition than the general population. The increased likelihood of developing a disease can be about a 10%, 20%, 50%, 100%, 150%, 200%, or greater increase.

[0049] The terms "treatment" and "therapy" as used herein refer to administering a drug or preparation to a clinically symptomatic individual suffering from a deleterious condition, disorder, or disease, so as to affect the reduction of the severity and / or frequency of symptoms, to eliminate symptoms and / or their underlying causes, and / or to facilitate the amelioration or repair of damage. It is understood that treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated, although it is not excluded.

[0050] In some cases, the compositions of the present invention are administered orally or systemically. Other modes of administration include rectal, topical, intraocular, buccal, intravaginal, intravesical, intracerebroventricular, intratracheal, nasal, transdermal, intra / on implant, or parenteral routes. The term "parenteral" includes subcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal, or injection. The compositions, including the compositions of the present invention, can be added to physiological fluids, such as blood. Oral administration may be preferred for preventive treatments due to patient convenience as well as dosing schedules. Parenteral modes (subcutaneous or intravenous) may be preferred for more acute illnesses or for treatment in patients who cannot tolerate enteral administration due to gastrointestinal intolerance, ileus, or other complications of severe illness. Inhalation therapy may be most appropriate for pulmonary vascular diseases (e.g., pulmonary hypertension).

[0051] In some embodiments, the compositions of the present invention may be administered orally to a subject in need thereof in the form of a capsule or tablet, hi some embodiments, the compositions of the present invention may be administered parenterally to a subject in need thereof in the form of a liquid.

[0052] The pharmaceutical composition can be assembled into a kit or pharmaceutical system for use in arresting the cell cycle in rapidly dividing cells, such as cancer cells. The kit or pharmaceutical system according to this aspect of the invention comprises a carrier means, such as a box, carton, or tube, having sealed therein one or more container means, such as a vial, tube, ampoule, bottle, syringe, or bag. The kit or pharmaceutical system of the invention may also include associated instructions for using the kit.

[0053] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0054] Where applicable or unless specifically disclaimed, it is contemplated that any one of the embodiments described herein can be combined with any other one or more embodiments, even if the embodiments are described under different aspects of the invention.

[0055] These and other embodiments are disclosed and / or encompassed by the following detailed description. [Brief description of the drawings]

[0056] [Figure 1A-1D] Figure 1A-D are a set of graphs, western blots, and heat maps showing that E1A binding protein (EP300), but not CREB binding protein (CBP), is required for neuroblastoma cell proliferation. Figure 1A is a heat map of the probability of dependency for neuroblastoma cell lines (n=19) in the DepMap 20Q2 data release. Figure 1B is a set of western blots of Kelly cells stably expressing Cas9 infected with single guide RNAs (sgRNAs) targeting EP300 (EP300-1,2), CBP (CBP-1,2) or controls (ch2.2, LACZ). Figure 1C is a graph of colony formation assays performed following sgRNA infection as in Figure 1B in Kelly and BE2C cells. n=3 independent replicates per cell line and treatment. *p<0.05. FIG. 1D is a graph of Kelly NB cells treated with a range of concentrations of EP300 / CBP complex inhibitors C646, CBP30, and A485 in a colony formation assay. n=3 independent replicates per cell line per treatment. See also FIG. 8A-FIG. 8N. [Figures 2A-2I]Figure 2A-I are a set of heatmaps, chromatin immunoprecipitation sequencing (ChIP-seq) tracks, and western blots and graphs showing that EP300 regulates the neuroblastoma core regulatory circuit directed by transcription factor activating protein 2B (TFAP2β). Figure 2A is a STRING database interaction plot of nuclear-dependent genes in neuroblastoma (NB) cells. Data are from Durbin et al. Nat Genet 50:1240-1246 (2018). Core regulatory circuit members (blue), or proteins with enzymatic domains (red), are shown. Figure 2B is a heatmap of ChIP-seq of EP300 and CBP in BE2C NB cells. Heatmap analysis was performed and ranked by the ratio of EP300:CBP binding. Data are representative of two cell lines, Kelly and BE2C. Figure 2C is a genome-wide heatmap analysis of Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and ChIP-seq of EP300, H3K27ac, and core regulatory circuit factors at binding sites of core transcriptional regulatory circuit (CRC) translation factor binding sites in BE2C and NB cells, ranked by MYCN binding. Data are representative of two cell lines, Kelly and BE2C. Figure 2D is a representative set of ChIP-seq tracks showing binding of CRC factors (blue), CBP (green), and EP300 (red) at the HAND2 locus in Kelly NB cells. Cardiac and neural crest derivatives expressed 2 (HAND2) super-enhancer (H3K27ac) and open chromatin (ATAC-seq) (black) are also shown. Data are representative of both Kelly and BE2C cells. Figure 2E is a graph of ChIP-seq motif enrichment analysis of EP300 and CBP in Kelly NB cells. Data was restricted to the top 500 binding peaks by EP300 or CBP in Kelly NB cells. Colored dots indicate known enriched transcription factors. Arrows indicate enriched motifs corresponding to TFAP2β.Figure 2F is a position-weight matrix from the analysis in Figure 2D showing the top enriched sequences under the EP300 peak compared to the CBP peak, which corresponds to the consensus binding sequence for TFAP2β. Figure 2G is a Western blot of co-immunoprecipitation of EP300 and CBP in Kelly NB cells. WCL = whole cell lysate. IgG = isotype-matched rabbit IgG antibody. Data are representative of three independent Western blots. Figure 2H is a Western blot of Kelly NB cells expressing Cas9 infected with sgRNA targeting TFAP2β (TFAP2β-1,2) or a control locus (ch2.2, LACZ). Data are representative of three independent lysates and blots. FIG. 2I is a graph of propidium iodide flow cytometry of Kelly NB cells expressing Cas9 and infected with sgRNA targeting TFAP2β (TFAP2β-1,2) or a control locus (ch2.2, LACZ). n=3 independent infections and flow analyses. *p<0.05. See also FIG. 9A-FIG. 9K. [Figures 3A-3I]Figures 3A-I are a set of chemical structures, graphs, and Western blots showing that JQAD1 is a selective EP300 degrader. Figure 3A is an image of the chemical structures of (R,S)-A485 and (R,S)-JQAD1 highlighting the structural components of JQAD1. Figure 3B is a graph of a CellTiter-Glo® assay of Kelly cells treated with 1 μM of (R,S)-A485, (R,S)-JQAD1, (S,S)-JQAD1 or dimethyl sulfoxide (DMSO) for 6 days. n=3 independent experiments and measurements at each time point. Figure 3C is a graph of an AlphaLISA® assay of multiple immunomodulatory imide drug (IMiD)-containing molecules (pomalidomide, thalidomide, lenalidomide, and JQAD1) and A485. Data are representative of three independent assays. FIG. 3D is a Western blot of Kelly cell lysates treated with a combination of biotin-JQAD1 or pomalidomide prior to streptavidin-bead purification. WCL = total cell lysate. Data are representative of three independent experiments and blots. FIG. 3E is a set of Western blots of Kelly NB cells treated for 24 hours (h) with DMSO, A485 or JQAD1 at the indicated concentrations (μM) prior to lysis. Data are representative of three independent experiments and blots. FIG. 3F is a graph of stable isotope labeling with amino acids in cell culture (SILAC)-labeled Kelly NB cells treated with 500 nM JQAD1 or DMSO vehicle for 24 hours prior to nuclear extraction and analysis by mass spectrometry. The ratio of peptides detected at 0 hours versus 24 hours is shown. Data represent the combined ratio of heavy:light labeled proteins detected in triplicate at 24 hours compared to 0 hours. The dotted line indicates a p-value of 0.01. Red dots indicate EP300 and CBP. n=3 independent treatments, lysates, and mass spectrometry reactions. Figure 3G is a Western blot of Kelly NB cells treated with 500 nM JQAD1 at the indicated time points before lysis. Data are representative of three independent experiments and blots. Figure 3H is a graph of propidium iodide (PI)-flow cytometry of subG1 events in Kelly (H) cells treated with JQAD1 or A485.Data are summary of n≧3 independent flow experiments. Similar results were obtained in SIMA cells treated with 1 μM of compound. Figure 3I is a graph of PI-flow cytometry of subG1 events in Kelly (H) and NGP (I) cells treated with JQAD1 or A485. Data are summary of n>3 independent flow experiments. See also Figures 10A-N. [Figure 4A-4E]Figure 4A-E are a set of western blots and plots showing that JQAD1, but not A485, disrupts chromatin localization of MYCN leading to apoptosis. Figure 4A is a western blot of markers of apoptosis cleaved caspase-3 and cleaved PARP1 in Kelly NB cells treated with 1 μM JQAD1, A485 or DMSO control for 12, 24 and 36 hours before lysis. Actin is shown as a loading control. Data are representative of three independent treatments and analyses in Kelly and NGP cells. Figure 4B is a plot of gene set enrichment analysis of RNAseq. Kelly cells were treated with 500 nM JQAD1, A485 or DMSO control for 24 hours prior to spike-in controlled by the External RNA Controls Consortium (ERCC) in RNAseq. Gene set enrichment analysis of RNAseq results was performed using the MSigDB Hallmarks dataset. n=3 biological replicates and independent RNA extractions per treatment. Figure 4C is a plot of normalized RNAseq gene expression of pro- and anti-apoptotic mRNA transcripts in Kelly cells treated with 500 nM JQAD1, A485 or DMSO control for 24 hours. Log10 transcript abundance normalized to DMSO and ERCC control is shown. n=3 biological replicates and independent RNA extractions per treatment. Figure 4D is a western blot of nuclear lysates of Kelly cells prepared and immunoprecipitated with anti-EP300, anti-CBP or IgG control antibodies. WCL=whole cell lysates. Data are representative of >3 independent co-immunoprecipitations and western blots. Figure 4E is a western blot of Kelly cells treated with DMSO control, A485 (0.5, 1 μM) or JQAD1 (0.5, 1 μM) followed by chromatin extraction. Total H3 is shown as a loading control. Data are representative of 3 independent treatments, lysates, and blots. [Figure 5A-5D]Figure 5A-D are a series of plots and ChIP-seq tracks showing that JQAD1 caused loss of histone H3K27 acetylation primarily at super-enhancers. Figure 5A is a set of plots of enhancers ranked by H3K27ac signal at 0 h (left) and 24 h (right) after treatment of Kelly cells with 500 nM JQAD1. Data are representative of two independent treatment and ChIP-seq experiments. Figure 5B is a series of plots of Log2 fold change of enhancer H3K27ac signal resolved by H3K27ac ChIP-seq in Kelly NB cells at 0 vs. 6 h (left) and 0 vs. 24 h (right). Data are normalized to Drosophila melanogaster outer chromatin. FIG. 5C is a plot of Log2 fold change of enhancer H3K27ac signals stratified by super-enhancers and typical enhancers at 6 and 24 hours after treatment of Kelly cells with 500 nM JQAD1. *** indicates p<0.0001 by Student's t-test comparing super-enhancer and typical enhancer regulated genes at 24 hours. FIG. 5D is a set of representative gene tracks of Kelly cells treated with 500 nM JQAD1 for 0 and 24 hours at the HAND2 core regulatory circuit factor locus. Data are representative of adrenergic CRC factor loci (HAND2, ISL1, PHOX2B, GATA3, TBX2, ASCL1 and TFAP2E) and two independent treatment and ChIP-seq experiments. See also FIG. 11A-B. [Figures 6A-6G]Figure 6A-6G are a series of plots and immunohistochemistry images showing that JQAD1 caused tumor growth inhibition and loss of EP300 in vivo. Figure 6A is a plot of Kelly NB xenografts established in NOD scid gamma (NSG™) mice and mice treated with vehicle control (n=11) or 40 mg / Kg JQAD1 daily (n=12) or twice daily intraperitoneally (IP). **p<0.01, ***p<0.001 by two-way analysis of variance (ANOVA) with post-hoc Tukey test. The kinetics of tumor growth curves were also analyzed by two-way ANOVA with mixed effects analysis. Figure 6B is a plot of Kaplan-Meier survival analysis of the mice described in Figure 6B, where 40 mg / Kg JQAD1 administered IP twice daily extended survival time compared to vehicle with log-rank test p<0.0001 for both JQAD1 treatment groups. Figure 6C is a plot of the body weight of the mice described in Figures 6B-6D. Figure 6D is a set of immunohistochemistry images of EP300 and CBP in Kelly cell xenografts treated for 14 days with either vehicle control or JQAD1 (40 mg / Kg, IP, daily). Data are representative of 3 independent animals per treatment. Bars = 50 μm. FIG. 6E is a plot of ERCC-spike in RNA-seq performed on tumor cells recovered from animals treated as described in FIG. 6D, showing fold change in expression in animals treated daily with 40 mg / Kg JQAD1 on day 14 (n=3) compared to vehicle control (n=4). RNA-seq groups of genes are stratified by regulation by typical or super enhancers, and gene identity of transcription factors or CRC genes. ***p<0.0001, *p=0.0223, ***p=0.0013 compared to CRC gene expression. See also FIG. 12A-D. [Figure 7A-7F]Figure 7A-7F are a series of plots and western blots showing that cancer cells showed biased dependence on EP300 compared to CBP. Figure 7A is a plot comparing the probability of dependence of EP300 and CBP in DepMap (n=757, 20Q2 release). *** indicates p<0.0001 by two-tailed Student's t-test. Figure 7B is a set of plots of the mean EP300 (black) and CBP (red) probability of dependence for the indicated cell lines. Figure 7C is a plot of the area under the curve (AUC) of the dose-response relationship for barcoded cancer cell lines (n=557) treated for 5 days with a range of concentrations of (R,S)-JQAD1. Cell lines were individually grouped into lineages. Red bars=median, individual black dots=individual cell lines, red dots=neuroblastoma cell lines. AUC was calculated from triplicate measurements at each dose at time=120 hours. Figure 7D is a plot of AUC values ​​of JQAD1 from Figure 7C against CRBN expression from Cancer Cell Line Encyclopedia (CCLE). *** p<0.001 by ANOVA for >5 TPM compared to <4 and 4-5 TPM with post-hoc Bonferroni correction. Figure 7E is a Western blot of BE2C cells stably expressing control (zsGreen) or CRBN (CRBN) treated with DMSO or 10 μM JQAD1 for 24 hours. Cell lysates were subjected to Western blotting for EP300, CBP and CRBN. Actin is shown as a loading control. Data are representative of three independent treatments and analyses. Figure 7F is a plot of BE2C cells stably expressing control (zsGreen) or CRBN (CRBN) treated with DMSO or 10 μM JQAD1 for 6 days prior to Cell-Titer Glo® analysis for cell proliferation. Data was normalized to BE2C-zsGreen, DMSO-treated cells. ***p=0.008 by Student's t-test comparing BE2C-CRBN DMSO-treated cells with JQAD1-treated cells. n=3 biological replicates. [Figures 8A-8N]Figure 8A-8N are a set of western blots and plots showing that EP300 and CBP expression are highly correlated in neuroblastoma and EP300 is required for neuroblastoma cell survival. Figure 8A is a western blot of BE2C cells expressing Cas9 infected with sgRNAs targeting EP300 (EP300-1,2), CBP (CBP-1,2) or control (ch2.2, LACZ). Data are representative of three independent sgRNA infections and lysates. Figure 8B is a plot of EP300 and CBP mRNA expression identified in primary neuroblastoma tumor samples. n=649, data retrieved from the R2 Genomics Browser, Kocak Neuroblastoma dataset (Kocak et al. Cell Death Dis 4:e586 (2013)). Figure 8C is a western blot of EP300, CBP and CRBN expression in neuroblastoma cell lines. Total H3 is shown as a loading control. Data are representative of three independent lysates. Figure 8D is a plot of mass spectrometry derived protein expression of EP300 and CBP retrieved from the DepMap portal as described in Nusinow et al. Cell 180:387-402 (2020). n=375 cancer cell lines. Figure 8E is a plot (TPM) of expression of CBP and EP300 (n=1371) across the cancer cell line encyclopedia as described in Ghandi et al. Nature 569:503-8 (2019). Figures 8F-H are a set of plots of colony formation assays of BE2C (Figure 8F), NB69 (Figure 8G) and NGP (Figure 8H) cells treated with various concentrations of combined EP300 / CBP inhibitors C646, CBP30 and A485. n=3 independent treatments per data point. Figures 8I-K are a series of plots of PI-flow cytometry analysis of BE2C (Figure 8I), Kelly (Figure 8J), and NGP (Figure 8K) cells treated for 24 hours with DMSO, A485, C646, or CBP30. n=3 independent treatments per cell line. *p<0.05. A485 was used at 500 nM (Kelly), 1 μM (BE2C, NGP).CBP30 was used at 1 μM (Kelly, BE2C), 2.5 μM (NGP). C646 was used at 2.5 μM (Kelly), 5 μM (BE2C, NGP). Figure 8L-Figure 8M are a series of plots of PI-flow cytometry analysis of Kelly (Figure 8L) and BE2C (Figure 8M) cells expressing Cas9 infected for 5 days with lentiviruses expressing sgRNAs targeting EP300 (EP300-1,2), CBP (CBP-1,2) or control (ch2.2, LACZ). n=3 independent infections and flow analyses. *p<0.05. Figure 8N is a western blot of BE2C and Kelly NB cells treated for 7 days with DMSO or A485 (BE2C 1 μM, Kelly 500 nM) prior to lysis. Data are representative of three independent lysates and blots. [Figure 9A-9K]Figure 9A-K are a set of heatmaps, plots, ChIP-seq tracks, and western blots showing that EP300 is recruited to the CRC locus through a physical interaction with the CRC member TFAP2β. Figure A is a heatmap of ChIP-seq of EP300 and CBP in Kelly NB cells (ranked by the ratio of EP300:CBP binding). Data are representative of two cell lines, Kelly and BE2C. Figure 9B is a genome-wide heatmap of ATAC-seq and ChIP-seq of EP300, H3K27ac, and core regulatory circuit factors at binding sites of core transcriptional regulatory circuit (CRC) translation factor binding sites in Kelly NB cells, ranked by MYCN binding. Data are representative of two cell lines, Kelly and BE2C. Figure 9C is a set of ChIP-seq tracks from Kelly cells showing binding of EP300 and CBP at core regulatory circuit loci and other neuroblastoma-associated loci marked by typical enhancers (upper panel) and super-enhancers (lower panel). Data are representative of Kelly and BE2C cells. Figure 9D is a plot of area under the curve analysis of motifs enriched under the top 500 unique EP300 and CBP peaks in BE2C cells. Arrows indicate motifs corresponding to TFAP213 that are enriched under the EP300 peak in both Kelly and BE2C cell lines. Figure 9E is a set of position weight matrices of enriched sequences corresponding to known transcription factors in the area under the ROC curve (AUROC) analysis. Figure 9F is a plot of the distribution of 35 proteins identified in both Kelly and BE2C cells after co-immunoprecipitation and mass spectrometry of H3K27ac from nuclear lysates. Normal rabbit IgG was immunoprecipitated as a negative control. Data represent proteins identified in both Kelly and BE2C cells using two independent co-IP-mass spectrometry reactions each. Figure 9G is immunoprecipitation-western blot data for EP300 and CBP in Kelly NB cells. WCL = whole cell lysate. IgG = isotype-matched rabbit IgG control. Data are representative of three independent blots.Figure 9H is a western blot of Kelly NB cells expressing Cas9 infected with lentivirus expressing sgRNAs against TFAP213 (TFAP213-1-4) or a control locus (ch2.2, LACZ). Data are representative of three independent blots. Figures 9I-9J are a set of western blots of Kelly NB cells expressing Cas9 infected with lentivirus expressing sgRNAs against GATA3 (GATA3-1,2) (Figure 9I), HAND2 (HAND2-1,2) (Figure 9J), or a control locus (ch2.2, LACZ). Data are representative of three independent blots. Figure 9K is a PI-flow cytometry plot of NGP cells expressing Cas9 infected with lentivirus expressing sgRNAs against TFAP213 (TFAP213-1,2) or a control locus (ch2.2, LACZ). n=3 independent infection and PI flow analyses. *p<0.05 compared to control. [Figures 10A-10N]Figures 10A-N are a series of plots and western blots showing that (R,S)-JQAD1 is a selective CRBN-dependent EP300 degrader that inhibits NB cell proliferation. Figures 10A-C are a series of plots of dose-viability measurements of Kelly (Figure 10A), NGP (Figure 10B) and SIMA (Figure 10C) NB cells in response to (S,S)-JQAD1, (R,S)-JQAD1 and (R,S)-A485 after 6 days. Data were resolved by Cell-Titer Glo® assay with n=3 independent measurements per time point and dose. Figure 10D-F are a series of plots of growth curves of Kelly (Figure 10D), NGP (Figure 10E) and SIMA (Figure 10F) NB cells in response to DMSO, (S,S)-JQAD1, (R,S)-JQAD1 and (R,S)-A485 over 6 days of treatment. Compound doses used were 1 μM (Kelly), 2.5 μM (NGP) and 1 μM (SIMA). Data were resolved by Cell-Titer Glo® assay with n=3 independent measurements per time point and dose. Figure 10G is a Western blot of Kelly NB cells treated with (R,S)-JQAD1 and (S,S)-JQAD1 for 24 hours before lysis. Data are representative of three independent Western blots. Figure 10H is a time course Western blot of NGP and SIMA cells treated with JQAD1 for 0-48 h before lysis. (R,S)-JQAD1 was used at 2.5 μM (NGP) and 1 μM (SIMA). Data are representative of three independent Western blots. * indicates cleaved poly[ADP-ribose] polymerase 1 (PARP1). Figure 10I is a Western blot of Kelly cells stably expressing Cas9 that were infected with sgRNA targeting CRBN (CRBN-1,3) or control loci (ch2.2, LACZ) and pools of knockout cells were established. Western blotting was performed with an antibody against CRBN. Data are representative of three independent Western blots.Figure 10J-K are a series of plots of CellTiter-Glo® assays of Kelly-Cas9 control or CRBN knockout cells treated for 7 days with a range of doses of JQAD1 (Figure 10J) or A485 (Figure 10K). n=3 independent replicates / dose and time point. Figure 10L is a Western blot of Kelly-Cas9 control (ch2.2, LACZ) and CRBN knockout cells treated with DMSO or 500 nM of (R,S)-JQAD1 for 24 hours. Data are representative of three independent treatments and lysates. * indicates cleaved PARP1. FIG. 10M is a Western blot of Kelly cells pretreated with DMSO, A485 (10 μM), pomalidomide (20 μM), bortezomib (2.5 nM), or MLN4924 (1 μM) for 4 hours followed by treatment with DMSO or JQAD1 (500 nM) for 48 hours. Data are representative of three independent blots. FIG. 10N is a set of PI flow cytometry plots of G1, S, and G2 / M phases in Kelly (top), NGP (middle), and SIMA (bottom) cells treated with DMSO (white), (R,S)-A485 (blue), or (R,S)-JQAD1 (red) for 0, 24, and 48 hours. Treatments were 500 nM (Kelly), 1 μM (NGP), and 1 μM (SIMA). Data are a summary of n≧3 independent flow experiments. ***q<0.001, **q<0.01, *q<0.05 compared to 0-hour controls by ANOVA with post-hoc multiple comparison correction. [Figure 11A-11B]Figure 11A-B are a set of ChIP-seq tracks and western blots showing that JQAD1 caused apoptosis and loss of enhancer acetylation at core regulatory circuit loci. Figure 11A is a western blot of NGP NB cells treated with 2.5 μM JQAD1, A485 or DMSO control for 12, 24 and 36 hours. Data are representative of three independent treatments and analyses. Figure 11B is a representative set of gene tracks of Kelly cells treated with 500 nM (R,S)-JQAD1 for 0 and 24 hours at core regulatory circuit factor loci. Data are representative of two independent treatments and ChIP-seq experiments. [Figure 12A-12D] Figure 12A-D are a series of plots and immunohistochemistry images showing that JQAD1 is effective in vivo with limited toxicity. Figure 12A is a plot of the pharmacokinetic analysis of JQAD1 after a single intraperitoneal dose at 10 mg / Kg in CD1 mice. Half-life=13.3 (+ / -3.37), Cmax=7 μM. n=3 mice. Figure 12B is a plot of daily dosing of increasing doses of JQAD1 in CD1 mice, which did not affect the weight of the animals. n=3 animals / group, serial weight measurements. Figure 12C is a plot of Balb / c CRBNILE391VAL humanized knock-in mice treated with vehicle or JQAD1 daily at 40 mg / Kg IP for 21 days and had their weight measured daily (n=3 mice per treatment group). Figure 12D is a set of immunohistochemistry images of formalin-fixed mouse liver tissue sections stained with hematoxylin and eosin to evaluate the possible toxicity and the effect of JQAD1 on the expression levels of EP300 and CBP. Three mice per treatment group were sacrificed 14 days after treatment. Data are representative of three independent animals per treatment (vehicle, JQAD1). Bar = 50 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] The present invention is based on the surprising discovery that E1A binding protein (EP300), but not its paralog CREB binding protein (CBP), is required for regulation of key enhancers in high-risk neuroblastoma (NB). EP300 is enhancer-regulated dependent in NB and is recruited to DNA via interaction with transcription factor activating protein 2B (TFAP2β), a member of the lineage-determination core regulatory circuitry in high-risk NB. Pharmacological degradation of EP300 by the proteolysis targeting chimera (PROTAC®) JQAD1 resulted in a global loss of histone acetylation in high-risk NB. Degradation of EP300 drove apoptosis due in part to loss of MYCN chromatin localization, with limited toxicity to non-transformed cells. Functional genomic and chemical analyses have revealed widespread dependence on EP300 in many types of human cancers, including myeloma, lymphoma, melanoma, rhabdomyosarcoma, colon, rectal, gastric, breast, brain, and pancreatic cancers.

[0058] High-Risk Neuroblastoma High-risk NBs are pediatric tumors of the peripheral sympathetic nervous system that originate from primitive neural crest cells and have poor survival rates. These neuroendocrine tumors are characterized by high expression of oncogenic MYC family members. (Matthay et al. Nat. Rev. Dis. Primers 2:16078(2016); Zimmerman et al. Cancer Discov. 8:320-35(2018)). MYCN is an essential member of a positive feed-forward autoregulatory loop of transcription factors (TFs) that establishes cell fate in MYCN-amplified NBs. This group of TFs is called the core regulatory circuit (CRC), and each member is regulated by a super-enhancer (SE) gene that is critically required for NB viability. One mechanism by which MYC family oncogenes drive tumor growth is by invading gene enhancers and recruiting transcriptional and epigenetic machinery (Zeid et al. Nat. Genet. 50:515-23 (2018)). Combined pharmacological inhibition of SE-mediated transcription initiation and elongation has been shown to rapidly disrupt NB CRC in vitro and in vivo, leading to transcriptional collapse and apoptosis (Durbin et al. Nat. Genet. 50:1240-6 (2018)).

[0059] Despite the fact that mass screening for NB does not significantly improve patient prognosis, some success has been achieved in NB therapy in recent years (Arakwa et al. J. Pediatr. 165:855-7 (2014)). NB proliferates and responds differently to treatment in different subjects. NB is classified by the International Neuroblastoma Risk Group (INRG) classification system into one of four categories: very low risk, low risk, intermediate risk, or high risk. Patients with low-risk and intermediate-risk neuroblastoma have a good prognosis, with excellent 5-year survival rates of over 90%, while the prognosis of high-risk neuroblastoma (HR-NB), which is detected in approximately 60% of cases, remains unfavorable (Kholodenko et al. J. Immunol. Res. 2018:7394268 (2018)). The 5-year survival rate remains below 50% despite aggressive multimodal therapy (Whittle et al. Expert Rev. Anticancer. Ther. 17:369-86 (2017)). Standard methods of neuroblastoma treatment have strong side effects, including severe damage to internal organs, anemia, effects on fertility, and hair loss. Chemotherapy, radiotherapy, and surgical methods show particularly low efficacy in the later stages of disease treatment, and they do not solve the problem of minimal residual disease, which is the cause of subsequent recurrence (Kholodenko et al. J. Immunol. Res. 2018:7394268 (2018)).

[0060] EP300 and CBP EP300 and CBP are paralogous multidomain protein acetyltransferases with broad cellular functions mediated by protein-protein interactions and catalytic acetyltransferase activity (Dancy and Cole, Chem. Rev. 115:2419-52 (2015)). These proteins are independently mutated or translocated in various human cancers, and numerous studies have identified distinct but overlapping activities of these proteins in non-transformed cell types, including embryonic and hematopoietic stem cells as well as more differentiated fibroblasts and T cells (Kasper et al. Mol. Cell. Biol. 26:789-809 (2006); Liu et al. Nat. Med. 19:1173-7 (2013); Rebel et al. Proc.Natl. Acad. Sci. USA 99:14789-94 (2002); Sen et al. Mol. Cell. 73:684-98 (2019); Yao et al. Cell 93:361-72 (1998)). EP300 and CBP show overlapping, but distinct binding patterns across the genome, indicating that these proteins have only partial functional redundancy in transcriptional regulation (Martire et al. BMC Mol. Cell. Biol. 21:55 (2020); Ramos et al. Nucleic Acids Res. 38:5396-5408 (2010)). Due to the high degree of homology between these proteins, especially in the HAT and bromodomains, it has been difficult to design small molecule inhibitors that are selective for any one of these proteins. To this end, studies have demonstrated that EP300 exhibits synthetic lethality in cell lines in which CBP is mutationally inactivated (Ogiwara et al. Cancer Discov. 6:430-45 (2016)). However, both enzymes are expressed in most cell lines and primary tissues, making it difficult to distinguish the functions of these two proteins.

[0061] An exemplary EP300 amino acid sequence is provided in NCBI Accession No. NP_001349772, version NP_001349772.1, as shown below (SEQ ID NO:1). JPEG2024509887000002.jpg82156 JPEG2024509887000003.jpg90156

[0062] An exemplary EP300 nucleic acid sequence is provided in NCBI Accession No. NM_001362843, version NM_001362843.2, as shown below (SEQ ID NO: 2). JPEG2024509887000004.jpg146156 JPEG2024509887000005.jpg252155 JPEG2024509887000006.jpg208156

[0063] An exemplary CBP amino acid sequence is provided in NCBI Accession No. XP_011520683, version XP_011520683.1, as shown below (SEQ ID NO:3). JPEG2024509887000007.jpg29156 JPEG2024509887000008.jpg131156

[0064] An exemplary CBP nucleic acid sequence is provided in NCBI Accession No. XM_011522381, version XM_011522381.2, as shown below (SEQ ID NO: 4). JPEG2024509887000009.jpg102156 JPEG2024509887000010.jpg251154 JPEG2024509887000011.jpg251155 JPEG2024509887000012.jpg70156

[0065] Celeblon (CRBN) Human CRBN is a 442 amino acid E3 ubiquitin ligase with an apparent molecular weight of approximately 51 kDa. CRBN contains the N-terminal portion (237 amino acids from amino acids 81 to 317) of the ATP-dependent Lon protease domain that lacks the conserved Walker A and Walker B motifs, 11 casein kinase II phosphorylation sites, 4 protein kinase C phosphorylation sites, 1 N-linked glycosylation site, and 2 myristoylation sites.

[0066] CRBN is widely expressed in the testis, spleen, prostate, liver, pancreas, placenta, kidney, lung, skeletal muscle, ovary, small intestine, peripheral blood leukocytes, colon, brain, and retina, and is localized in the cytoplasm, nucleus, and plasma membrane (e.g., peripheral membrane). (Chang et al. Int. J. Biochem. Mol. Biol. 2:287-94 (2011)). Cereblon is an E3 ubiquitin ligase that forms a complex with damaged DNA-binding protein 1 (DDB1), cullin-4A (CUL4A), and regulator of cullin 1 (ROC1). This complex also ubiquitinates many other proteins. Cereblon ubiquitination of target proteins results in increased levels of fibroblast growth factor 8 (FGF8) and fibroblast growth factor 10 (FGF10). FGF8 controls several developmental processes, such as the formation of limbs and auditory vesicles.

[0067] An exemplary CRBN amino acid sequence is provided in NCBI Accession No. XP_011532093, version XP_011532093.1, as shown below (SEQ ID NO:5). JPEG2024509887000013.jpg33161

[0068] An exemplary CRBN nucleic acid sequence is provided in NCBI Accession No. XM_011533791, version XM_011533791.3, as shown below (SEQ ID NO: 6). JPEG2024509887000014.jpg251157

[0069] EP300 is required for high-risk NB proliferation. High-risk neuroblastoma requires a group of 147 genes for survival (Durbin et al. Nat. Genet. 50:1240-6 (2018)). One of these genes is the histone acetyltransferase enzyme EP300, but not its paralog CBP, which is surprising since EP300 often overlaps with CBP (Dancy and Cole, Chem. Rev. 115:2419-52 (2015)). Both EP300 and CBP acetylate the Lys-27 residue of histone H3 (H3K27ac), a mark associated with active gene transcription (Dancy and Cole, Chem. Rev. 115:2419-52 (2015); Durbin et al. Nat. Genet. 50:1240-46 (2018)). EP300, interestingly, appeared to be specifically required in neuroblastoma compared to CBP. Therefore, we investigated the relative expression and dependency of these two genes across a panel of representative neuroblastoma cell lines. First, the relative dependency of EP300 or CBP was examined in 19 high-risk neuroblastoma cell lines using the DepMap exome-wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) deletion dataset (Meyers et al. Nat. Genet. 49:1779-84 (2017)). Examination of possible dependency on EP300 and CBP in this panel of neuroblastoma cell lines demonstrated that the majority of the cell lines required EP300 for cell proliferation (Figure 1A). Interestingly, in four of the cell lines with high levels of dependency on EP300, dependency on CBP was also observed, indicating that each protein is essential for driving the expression of a distinct subset of genes (Figure 1A). An additional four cell lines were not dependent on either EP300 or CBP, potentially indicating redundancy of these two acetyltransferases in these cell lines (Figure 1A).To extend these findings, CRISPR-Cas9-mediated knockout of EP300 and CBP was performed in two MYCN-amplified NB cell lines, Kelly and BE2C (Figure 1B, Figure 8A). Although both EP300 and CBP are highly expressed in these two cell lines, loss of EP300 caused a marked loss of H3K27ac expression, whereas loss of CBP had a minimal effect, similar to the control single guide RNA (sgRNA), indicating that most enhancers and promoters depend on EP300 to catalyze H3K27ac in these cell lines. Furthermore, expression of MYCN, a well-known dependency in MYCN-amplified neuroblastomas, was almost entirely dependent on EP300 and independent of CBP (Figure 1B, Figure 8A). Thus, CRISPR-Cas9 inactivation of EP300, but not CBP inactivation, significantly reduced colony formation in each cell line (Figure 1C). The remaining colonies formed by the EP300 CRISPR knockout cells did not express green fluorescent protein (GFP) co-expressed in the vector containing the guide RNA, indicating that they represented cells that were not infected with the vector containing the EP300-targeting guide RNA.

[0070] Analysis of EP300 and CBP messenger RNA (mRNA) expression in primary neuroblastoma tumors revealed a positive correlation in expression levels (Figure 8B). Furthermore, analysis of publicly available sequencing data in primary neuroblastoma tumors demonstrated that mutations in EP300 or CBP are rare in human high-risk neuroblastomas, including inactivating mutations (e.g., nonsense or frameshift mutations) (Zhou et al. Nat. Genet. 48:4-6 (2016)). By western blotting, EP300 and CBP levels were generally similar to each other across a panel of neuroblastoma cell lines (Figure 8C). Analysis of cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE) proteomics and mRNA expression datasets also showed correlated expression levels of EP300 and CBP at both the RNA and protein levels, including cell lines derived from patients with neuroblastoma (red), indicating that these findings are relevant across multiple tumor lineages (Figures 8D-E).

[0071] To test the genetic findings using small molecule probes, we next performed colony formation assays of NB cells with known combination inhibitors of EP300 and CBP, including two inhibitors targeting the HAT domains-A485 and C646, and one inhibitor targeting the bromodomain-CBP30 (Lasko et al. Nature 550:128-32 (2017); Yan et al. J. Invest. Dermatol. 133:2444-52 (2013); Hammitzsch et al. Proc. Natl. Acad. Sci. USA 112:10768-73 (2015)). These inhibitors are known to be non-selective between the two HATs. Across multiple NB cell lines, the most potent compound in reducing neuroblastoma colony formation was the HAT domain inhibitor A485 (Figure 1D, Figure 8F-Figure 8H). Combined inhibition of EP300 / CBP with A485 caused G1 cell cycle arrest within 24 hours (Figure 8I-K), similar to the effect of knockout of EP300 but not CBP (Figure 8L-M). Chronic treatment for 7 days resulted in a global loss of H3K27Ac modification, loss of MYCN expression, and induction of cleaved caspase 3 and poly[ADP-ribose]polymerase 1 (PARP1), indicative of apoptotic cell death (Figure 8N). Thus, both cell cycle progression and cell survival are impaired by inhibition of the HAT activity of both EP300 and CBP. Genetic studies showed that this is due to a dependency on EP300, but not CBP, for proliferation and survival of most MYCN-amplified neuroblastoma cell lines.

[0072] EP300 promotes NB adrenergic CRC-driven transcription through binding to TFAP2β. We next investigated the mechanism by which EP300, but not CBP, is required for the proliferation of MYCN-amplified neuroblastoma cell lines. Core regulatory circuit (CRC) transcription factors (TFs) have been identified as crucial for determining cell fate in neuroblastoma and are marked and regulated by extensive expansions of histone H3K27ac (Boeva ​​et al. Nat. Genet. 49:1408-13 (2017); Durbin et al. Nat. Genet. 50:1240-46 (2018); van Groningen et al. Nat. Genet. 49:1261-6 (2017)). This analysis revealed that master transcription factors in adrenergic subtype NBs include HAND2, ISL1, PHOX2B, GATA3, TBX2, and ASCL1. Therefore, we also investigated the mechanism by which EP300 cooperates with NB CRC-driven gene expression programs. An interaction analysis of all expressed nuclear neuroblastoma-dependent genes was performed using the Search Tool for the Retrieval of Interacting Genes / Proteins (STRING) database (Szklarczyk et al. Nucleic Acids Res. 43:D447-52 (2015)). This analysis demonstrated that EP300 was found in a densely interacting network of genes enriched for CRC transcription factors (Figure 2A) (Durbin et al. Nat. Genet. 50:1240-6 (2018); Wang et al. Nat. Commun. 10:5622 (2019)). To understand the genome-wide binding patterns of EP300 and CBP, we performed chromatin immunoprecipitation coupled to massively parallel high-throughput sequencing (ChIP-seq) in two distinct MYCN-amplified neuroblastoma cell lines, BE2C and Kelly, using antibodies that recognize H3K27ac, EP300, and CBP (Fig. 2B, Fig. S4A).This was also compared to Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) data from both cell lines. Genome-wide heat map analysis of binding demonstrated that EP300 exhibited a similar binding pattern with H3K27ac (Figure 2B, Figure 2A). In contrast, CBP binding showed a different pattern, enriched in regions most densely occupied by EP300, with many regions depleted of EP300 (Figure 2B, Figure 2A). This result indicates that EP300 binds primarily to active enhancers marked by H3K27ac and accessible to transposase by ATAC sequencing (Figure 2B, Figure 2A). Next, to examine the importance of EP300 in regulating gene expression programs in neuroblastoma cells, the degree of EP300 colocalization was assayed with previously defined targets of neuroblastoma CRC transcription factors (Figure 2C, Figure 9B). Genome-wide heatmap analysis ranked by MYCN binding demonstrated that EP300 exhibited similar binding patterns to each of the CRC TFs (Figure 2C, Figure 9B). Specific assessment of EP300 and CBP binding at specific CRC TF coding loci marked by high density H3K27ac signals demonstrated colocalization of EP300 with CRC transcription factors at sites bound by all CRC members (Figure 2D, Figure 9C, red tracks). In contrast, CBP was only minimally enriched at the loci of CRC transcription factors (Figure 2D, Figure 9C, green tracks). These data indicate that EP300, but not CBP, is preferentially localized across the genome at open chromatin sites densely enriched for binding of CRC factors, including enhancers that control the adrenergic CRC gene itself and sites within enhancers of genes that comprise the extended regulatory network of adrenergic CRC in NB cells (Durbin et al. Nat. Genet. 50:1240-6 (2018)).

[0073] Both EP300 and CBP lack sequence-specific DNA binding activity and require association with DNA-binding factors to achieve locus-specific binding (Song et al. Biochem. Biophys. Res. Commun. 296:118-24 (2002)). Therefore, we investigated the mechanism by which EP300 is targeted to chromatin loci associated with enhancers in CRC. To identify proteins involved in EP300 recruitment to DNA in NB cells, motif enrichment analysis of the top 500 peaks bound by either EP300 or CBP was performed in Kelly and BE2C NB cell lines. Consistent with previous evidence showing that EP300 protein forms interactions with several TFs to nucleate several structures that enhance higher order binding, this analysis demonstrated enrichment for several transcription factor consensus binding motifs that preferentially associated with either EP300 or CBP binding (Figures 2E-F, 9D-E) (He et al. Nucleic Acids Res. 39:4464-74 (2011)). Two motifs corresponding to consensus binding sequences for the transcription factors GATA3 and TFAP2β were selectively enriched under the EP300 binding peaks in both cell lines (Figures 2E-F, 9D-E). To validate that these transcription factors associate with H3K27ac-labeled chromatin, we next performed co-immunoprecipitation of H3K27ac in nuclear extracts of Kelly and BE2C cells, followed by mass spectrometry of isolated proteins. As expected, peptides corresponding to EP300 and CBP that co-immunoprecipitated with H3K27ac were detected in both Kelly and BE2C cells. However, this experiment also identified four transcription factors, including GATA3 and TFAP2β, that physically interacted with H3K27ac-labeled nucleosomes in both cell lines (Table 3, Example 5, and Figure 9F). GATA3 is a known member of CRC in high-risk NB cells (Boeva ​​et al. Nat. Genet. 49:1408-13 (2017); Durbin et al. Nat. Genet. 50:1240-6 (2018)).TFAP2β has been identified as a possible CRC member because it is a transcription factor dependent in NBs commonly regulated by super-enhancers (Boeva ​​et al. Nat. Genet. 49:1408-13 (2017); van Groningen et al. Nat. Genet. 49:1261-6 (2017); Durbin et al. Nat. Genet. 50:1240-6 (2018)). Using a new antibody against TFAP2β, we performed ChIP-seq for TFAP2β binding, and the results show that TFAP2β binds to super-enhancers and regulates other members of the CRC (Figures 2B-C, 9B-C). Thus, TFAP2β is a newly identified member of the adrenergic core regulatory circuit in MYCN-amplified NB cells.

[0074] Because EP300 binding was enriched at sites containing GATA3 and TFAP2β motifs, and all of these proteins bound to H3K27ac-labeled chromatin, we then investigated the physical association of EP300 with GATA3 and TFAP2β. Immunoprecipitation of EP300 and CBP in Kelly NB cells, followed by western blotting for TFAPβ and GATA3, demonstrated that EP300, but not CBP, physically interacted with both TFAPβ and, to a lesser extent, GATA3 (Figure 2G, Figure 9G). Furthermore, reciprocal co-immunoprecipitation of TFAP2β in Kelly cells demonstrated the presence of EP300 protein, but not CBP (Figure 9G). To determine whether these transcription factors could control EP300 localization, CRISPR-Cas9-based knockout of TFAP2β or GATA3 was performed in Kelly NB cells (Figure 2H, Figure 9H-I). As a control, we also performed knockout studies of HAND2, a CRC factor that did not show selective motif enrichment under the EP300 peak (Figure 9J). Five days after knockout of TFAP2β, loss of TFAP2β and H3K27ac expression levels was observed without any effect on the expression levels of EP300 or CBP (Figure 2H, Figure 9H). In contrast, knockout of either GATA3 or HAND2 did not affect the levels of H3K27ac (Figure 9I-Figure 9J). Similar to knockout of EP300, knockout of TFAP2β also led to G1 cell cycle arrest in Kelly and NGP NB cells (Figure 2I, Figure 9K). Thus, these data indicate that EP300 is targeted to DNA via physical interaction with the CRC transcription factor TFAP2β in neuroblastoma.

[0075] EP300 was selectively degraded by the proteolytic targeting chimera (PROTAC®), JQAD1. All small molecules currently available that inhibit the HAT activity of EP300 have similar K dand inhibit the HAT activity of CBP (Dancy and Cole, Chem. Rev. 115:2419-52 (2015); Hammitzsch et al. Proc. Natl. Acad. Sci. USA 112:10768-73 (2015); Lasko et al. Nature 550:128-32 (2017); Yan et al. J. Invest. Dermatol. 133:2444-52 (2013)). This includes A485, the most potent and specific HAT inhibitor developed to date (Lasko et al. Nature 550:128-32 (2017); Michaelides et al. ACS Med. Chem. Lett. 9:28-33 (2018)). One approach to selectively target EP300 in neuroblastoma could be to disrupt the interaction between TFAP2β and EP300, but such strategies are typically difficult to implement (reviewed in: Wimalasena et al. Mol. Cell. 78:1086-95 (2020)). Recently, an alternative approach to developing selective compounds has been shown to be the development of small molecule degraders called "PROTACs". PROTACs are heterobifunctional small molecules that bind to target proteins and mediate the formation of ternary complexes between the target proteins and E3 ligase receptors (reviewed in: Burslem and Crews, Cell 181:102-14 (2020)). The ternary complex formed by the PROTAC® and the target protein crosslinks to an E2 ubiquitin ligase, which polyubiquitinates the target protein and targets it to the proteasome for degradation and recycling (Burslem and Crews, Cell 181:102-14 (2020)). To this end, the degrader A485 has been reported to indiscriminately degrade EP300 and CBP using bait molecules that target the bromodomains of these proteins (Vannam et al. Cell Chem Biol, 28:503-14.e12 (2020)).However, A485 was the most potent small molecule inhibitor in neuroblastoma cells, with the lowest K for EP300 and CBP of all small molecules targeting these proteins. d Since A485 has value, we tested the activity of small molecule degraders using A485 as a bait molecule (Figure 3A) (Lasko et al. Nature 550:128-32 (2017)). Computer structural modeling of the interaction between the HAT domain of EP300 and the immunomodulatory imide drug (iMiD) binding region of CRBN indicated that the optimal linker length between A485 and E3 ligases is 8 to 12 carbons. Therefore, we designed and synthesized a compound, JQAD1, which contains the two chiral centers found within A485 and a 12-carbon linking chain (Figure 3A, Scheme 1) (Michaelides et al. ACS Med. Chem. Lett. 9:28-33 (2018)).

[0076] Three neuroblastoma cell types expressing high levels of CRBN, Kelly, NGP, and SIMA, were treated with purified (R,S) and (S,S) stereoisomers of JQAD1 (Figure 8C). By comparison, the (R,S) stereoisomer had the lowest IC in intact Kelly, NGP, and SIMA NB cells. 50 This IC has a concentration 50 is the IC of the parent molecule A485 50 It was established that JQAD1 is less potent than the (R,S) stereoisomer (Figure 3B, Figure 10A-F). Therefore, the term JQAD1, as used herein, refers to the more active (R,S) stereoisomer of a PROTAC® compound, unless otherwise indicated, with the (S,S) stereoisomer being used as the control.

[0077] To determine whether JQAD1 interacts with the E3 ligase receptor CRBN, an AlphaLISA® fluorescent assay was performed using biotinylated pomalidomide and His-tagged CRBN bound to beads using the AlphaLISA® platform (Yasgar et al. Methods Mol. Biol. 1439:77-98 (2016)). All IMiD-containing compounds, including JQAD1 and free pomalidomide, efficiently interacted with CRBN in the AlphaLISA® assay, whereas the parent compound A485 did not (Figure 3C). Next, biotinylated JQAD1 (biotin-JQAD1, Scheme 2) was synthesized and incubated with Kelly cell lysate, followed by purification with streptavidin. Western blotting of the purified lysate demonstrated the presence of EP300 and CRBN, but surprisingly, the CBP protein was absent (Figure 3D). Furthermore, co-treatment of Kelly cell lysates with JQAD1 and excess pomalidomide (to compete for binding to CRBN) resulted in partial loss of interaction between JQAD1, CRBN, and EP300, indicating that these three proteins form a ternary complex (Figure 3D). Surprisingly, the applicant discovered that the PROTAC® JQAD1 specifically binds to EP300, a major mediator of H3K27ac in high-risk neuroblastoma. PROTAC® may acquire preferential specificity for one of two possible target enzymes due to limited three-dimensional interactions, as described in Burslem and Crews, Cell 181:102-14 (2020). In this case, preferential targeting of EP300 by PROTAC® JQAD1 is clearly advantageous over conventional compounds, as neuroblastoma cells often rely exclusively on EP300, whereas normal cells of a different lineage may require CBP and are spared from toxicity by the specificity of PROTAC® JQAD1.

[0078] Because JQAD1 preferentially interacted with both EP300 and CRBN, we investigated whether JQAD1 preferentially induced degradation of EP300 compared to CBP in MYCN-amplified neuroblastoma cells. Treatment of Kelly cells for 24 hours (h) with JQAD1 demonstrated a dose-dependent decrease in EP300 expression with a parallel loss of H3K27ac modification (Figure 3E). Similar treatment of Kelly cells with A485 caused loss of H3K27ac due to catalytic inhibition of EP300 enzymatic activity (Figure 3E). Treatment of Kelly cells with (R,S)-JQAD1 and control (S,S)-JQAD1 for 24 h revealed that (S,S)-JQAD1 had limited effects on H3K27ac or EP300 expression levels, whereas (R,S)-JQAD1 suppressed both H3K27ac and EP300 expression levels (Figure 10G). Consistent with the specificity of JQAD1 for EP300, neither compound had a significant effect on the expression level of CBP at this time point (Figure 10G). To further examine the specificity of JQAD1 for EP300, we performed an analysis of the effect of JQAD1 in Kelly cells labeled with stable isotope labeling by amino acids in cell culture (SILAC) (Figure 3F). Kelly cells were cultured in SILAC medium containing heavy or light labeled arginine and lysine. Prior to nuclear extraction and protein lysis, heavy labeled cells were treated with 500 nM JQAD1 and light labeled cells were treated with dimethyl sulfoxide (DMSO) for 24 h. As a control, nuclear extraction and lysis were performed on untreated heavy and light labeled Kelly cells. Protein abundance was then analyzed by mass spectrometry to determine global changes in the nuclear proteome. After 24 h treatment with JQAD1, EP300 protein was significantly decreased (p=3.3×10 -5), CBP and other proteins in the nuclear proteome remained detectable at levels similar to controls (Figure 3F). To extend these findings, three NB cell lines with high levels of CRBN (e.g., protein or mRNA), Kelly, NGP and SIMA (Figure 8C), were treated with JQAD and the effects on specific proteins were measured by Western blotting. In all three cell lines, JQAD1 induced selective loss of EP300 expression by 24 hours, which coincided with cleavage of PARP1, signaling the onset of apoptosis (Figure 3G, Figure 10H). At this time point, CBP could still be detected in all three cell lines. With prolonged treatment, loss of CBP expression was observed, but this could not be separated from the general effects of apoptosis (Figure 3G, Figure 10H).

[0079] JQAD1 contains an IMiD moiety that interacts with the E3 ligase receptor CRBN (Figure 3C-D). To genetically demonstrate that CRBN is required for JQAD1-mediated EP300 degradation and cellular effects, CRISPR-Cas9 gene editing was used to generate Kelly cells with stable disruption of the CRBN gene. Western blotting of lysates prepared from control or CRBN-edited Kelly cells demonstrated retained expression in control-edited cells and loss of CRBN expression in CRBN-edited cells (Figure 10I). Control-edited Kelly cells were potently killed by JQAD1, whereas CRBN knockout cells were resistant to the effects of JQAD1, indicating that CRBN expression is required for JQAD1 growth-suppressing activity (Figure 10J). In contrast, A485 inhibited the proliferation of both CRBN knockout cells and controls equally (Fig. 10K), indicating that loss of CRBN does not affect the enzymatic function of EP300. Furthermore, Western blots of lysates prepared from control and CRBN knockout Kelly cells treated with JQAD1 or DMSO demonstrated that JQAD1 suppressed EP300 expression and H3K27ac modification and induced apoptosis indicated by PARP1 cleavage in control knockout cells but not in CRBN-edited cells (Fig. 10L). Thus, CRBN is required for JQAD1-mediated EP300 degradation and induction of apoptosis. As treatment of CRBN-edited cells with JQAD1 did not affect H3K27ac, the structure of JQAD1 prevents its A485 moiety from competitively inhibiting EP300 HAT activity, and thus acts as a pure CRBN-dependent proteolytic agent without catalytic inhibitory activity (Fig. 10L). To further investigate the pathways involved in JQAD1-mediated EP300 degradation, we performed western blots on Kelly cells co-treated with JQAD1 and other compounds predicted to disrupt JQAD1 function. EP300 degradation was inhibited by excess A485 or IMiD (pomalidomide), or by co-treatment with a proteasome inhibitor (bortezomib) or an E3 ubiquitin ligase neddylation inhibitor (MLN4924) (Figure S4M).These data indicate that JQAD1 functions by binding to EP300, leading to its CRBN-dependent proteasomal degradation.

[0080] JQAD1 resulted in a robust CRBN- and proteasome-dependent loss of EP300 and cell death. To assess the mechanism by which JQAD1 reduces cell proliferation, Kelly and NGP cells were treated with JQAD1, A485, or vehicle control and subjected to propidium iodide flow cytometry. Cells treated with A485 exhibited a G1 cell cycle arrest phenotype (Figure 10N). However, the use of (R,S)-JQAD1 to degrade EP300, but not the combined catalytic inhibition of both EP300 and CBP, resulted in the induction of a subG1 peak consistent with apoptotic cell death (Figure 3H; Figure 10I). Thus, while A485 slows cell proliferation by G1 cell cycle arrest, (R,S)-JQAD1 has its own effect by inducing apoptotic cell death.

[0081] JQAD1 dissociated the MYCN protein from chromatin. One important difference between the acute effects of A485 and JQAD1 treatment is that JQAD1 induced apoptosis consistent with the kinetics of loss of EP300, whereas A485 treatment led to G1 cell cycle arrest (Figures 3G-I, 10H-N). To further demonstrate this difference in the induction of apoptosis by these two drugs, Kelly and NGP cells were treated with equal concentrations of A485 or JQAD1 for 12-36 h prior to protein extraction to analyze the effect on apoptosis. Treatment of both cell lines with JQAD1 induced cell apoptosis characterized by cleavage of caspase-3 and PARP1. In contrast, treatment with A485 had little effect on these parameters (Figures 4A, 11A). To evaluate the mechanism underlying the difference in response, Kelly NB cells were treated with equivalent concentrations of DMSO, JQAD1 or A485 for 24 hours prior to extraction of total RNA. RNA samples were then normalized with exogenous spike-in RNA and used for RNA-seq analysis. RNA-seq results for JQAD1 and A485 treated samples were then compared by gene set enrichment (GSEA) analysis. Consistent with our flow cytometry studies, GSEA analysis of hallmark gene sets demonstrated enrichment of apoptosis hallmark gene sets in JQAD1 treated cells compared to A485 treated cells (Figure 4B).Furthermore, JQAD1-treated cells showed upregulation of proapoptotic BH3-only effectors B-cell lymphoma 2-like protein 11 (BIM), BH3 interacting-domain death agonist (BID), and p53-upregulated modulator of apoptosis (PUMA), along with the proapoptotic mediator BCL2 associated X protein (BAX) and its inhibitors BCL2 and myeloid leukemia 1 (MCL1), although the transcriptional levels of each of these mRNAs were not affected in A485-treated cells (Figure 4C). This suggests that these mRNA transcripts are upregulated via CBP, as JQAD1 specifically affected EP300, while A485 inhibited transcriptional activation by both HAT proteins.

[0082] One mechanism by which NB cells suppress apoptosis is through high expression and transcriptional activity of the MYCN oncoprotein, sometimes referred to as "oncogene addiction" (reviewed in: Gabay et al. Cold Spring Harb. Perspect. Med. 4:a014241 (2014); Huang and Weiss, Cold Spring Harb Perspect Med. 3:a014415 (2013)). In addition, EP300 and CBP are known to regulate the MYCN family member c-MYC protein through protein-protein interactions (Faiola et al. Mol. Cell Biol. 25:10220-34 (2005); Vervoorts et al. EMBO Rep. 4:484-90 (2003); Zhang et al. Biochem. Biophys. Res. Commun. 336:274-80 (2005)). Therefore, it is hypothesized that a similar physical interaction exists between MYCN and EP300, which may result in stabilization of MYCN expression. We therefore performed co-immunoprecipitation assays using antibodies targeting endogenous EP300 or CBP in Kelly NB cells. Immunoprecipitation of proteins from Kelly nuclear lysates with anti-EP300 antibodies, followed by Western blotting, demonstrated significant association with MYCN protein. In contrast, immunoprecipitation of CBP, as well as the IgG control, did not show any association with MYCN protein (Figure 4D). Thus, in Kelly NB cells, EP300, but not CBP, physically interacts with the MYCN oncoprotein. To assess the functional significance of this interaction, we treated Kelly NB cells with DMSO or various concentrations of A485 or JQAD1 for 24 hours, then isolated chromatin protein extracts to assess the presence of chromatin-bound MYCN. Kelly cells treated with A485 showed no MYCN loss from chromatin extracts up to 1.0 μM ( Fig. 4E ).In contrast, cells treated with JQAD1 to degrade EP300 showed loss of both EP300 and chromatin-bound MYCN proteins (Figure 4E). These data support the interpretation that EP300 degradation by JQAD1 results in loss of chromatin-bound MYCN, while inhibition of EP300 HAT activity does not affect chromatin localization. Thus, a physical interaction between EP300 and MYCN, separate from HAT enzymatic activity, keeps MYCN on chromatin, which is necessary to promote cell proliferation and suppress apoptosis in NB cells.

[0083] JQAD1 caused the loss of H3K27Ac enriched at super-enhancers. Because JQAD1 selectively degraded EP300 with minimal effects on CBP up to 48 h in NB cell lines, we used JQAD1 to assess the effects of EP300 loss on genome-wide H3K27ac modifications. To determine these effects, we performed ChIP-seq over a time course from 0 to 24 h after (R,S)-JQAD1 exposure using an antibody that recognizes H3K27ac in Kelly NB cells. These samples were externally normalized using spike-in Drosophila melanogaster chromatin. Comparison of all H3K27ac-labeled sites to untreated samples demonstrated an approximately two-fold overall repression of all enhancers by 24 h of treatment, a time point at which EP300 was degraded and CBP was retained (Figure 5A). Comparison of H3K27ac signals at earlier time points (6 h) with 0 h controls showed no consistent changes in acetylation, but by 24 h of treatment, there was a general loss of H3K27ac signals throughout the genome, which was most prominent at densely acetylated super-enhancers, including those that regulate core regulatory circuits (Figures 5B-D, 11B). These data indicate that super-enhancer loci in Kelly cells are primarily regulated by EP300 and not CBP, as at this time point EP300 was degraded without affecting levels of CBP protein expression (Figures 5D, 11B).

[0084] JQAD1 was effective with limited toxicity in vivo. As several CRBN-based PROTAC® agents have been shown to cause targeted protein degradation in vivo (reviewed in: Burslem and Crews, Cell 181:102-14 (2020)), we investigated whether JQAD1 actively degrades EP300 in vivo in a human neuroblastoma xenograft model. First, pharmacokinetic analysis following a single intraperitoneal administration of 10 mg / Kg of JQAD1 was performed to identify the half-life and maximum serum concentration of the compound. Following intraperitoneal administration of 10 mg / Kg, JQAD1 had a half-life of 13.3 (+ / - 3.37 SD) hours in mouse serum and a C of 7 μM. max (FIG. 12A), which indicates that IC 50 (Figure 8A-8C). The maximum tolerated dose (MTD) in a mouse model was then investigated. Increasing doses of JQAD1 were injected intraperitoneally daily. Daily intraperitoneal treatment with JQAD1 was well tolerated without signs of weight loss in the animals (Figure 12B). Of note, at doses of JQAD1 higher than 40 mg / Kg, precipitation of the compound was observed in the peritoneal cavity. Therefore, 40 mg / Kg of JQAD1 was determined to be the maximum dose of a single intraperitoneal administration without obvious toxicity in mice.

[0085] Subcutaneous xenografts of Kelly cells were then established into the flanks of NOD scid gamma (NSG™) mice, and the mice were treated intraperitoneally with either vehicle control or 40 mg / Kg JQAD1 once or twice daily (FIG. 6A). Both twice daily JQAD1 treatments, and to a lesser extent once daily treatments, caused suppression of tumor growth of the xenografts by the third day of treatment (p<0.05 by two-way ANOVA with post hoc Tukey test for tumor size, p<0.05 for suppressed growth rate in JQAD once daily or twice daily treated tumors by mixed effects analysis with post hoc Tukey's multiple comparison test) and prolonged survival (p<0.0001 by log-rank test for once and twice daily administration, respectively, compared to vehicle control) (FIGS. 6A-B). The effect of JQAD1 treatment on body weight was also monitored, with control animals maintaining their weight over 14 days of treatment before they began to require sacrifice due to tumor burden (Figure 6C). In a separate experiment, NSG™ mice were again xenografted with Kelly cells and treated with vehicle control or JQAD1 at 40 mg / Kg once daily for 10 days. Animals were then sacrificed and tumors excised. Tumor material was fixed for immunohistochemistry (IHC) and processed into single cells prior to RNA-seq analysis of External RNA Control Consortium (ERCC) controls (Figure 6D-E). Tumors recovered from animals treated with JQAD1 showed loss of EP300 immunostaining compared to vehicle control tumors, but CBP immunostaining was retained (Figure 6D). Furthermore, RNA expression profiles of tumor cells from mice treated with JQAD1 demonstrated preferential downregulation of genes regulated by super-enhancers compared to those regulated by typical enhancers, compared to vehicle controls (Figure 6E, p<0.0001).

[0086] Human CRBN is a Crbn analogue of mouse Crbn Ile391 Compared to the CRBN, the important amino acid residue Val388Unlike mice, JQAD1 is important for the binding, ubiquitination, and degradation of key substrates, including spalt-like transcription factor 4 (SALL4), a member of the spalt-like family of developmental transcription factors (Donovan et al. Elife 7:e38430 (2018); Fink et al. Blood 132:1535-44 (2018)). Therefore, to more rigorously evaluate the potential activity and toxic effects of JQAD1 on mouse tissues, we transfected Balb / c Crbn ILE391VAL Humanized knock-in mice were dosed daily for 21 days at 40 mg / Kg IP (Fink et al. Blood 132:1535-44 (2018)). JQAD1 at this dose was well tolerated and did not affect grooming, behavior, body weight, peripheral blood counts, liver function tests or creatinine measurements performed 14 days after treatment (Table 1, Figure 12C). After 14 days of treatment, three mice per each treatment group were sacrificed and the skin, brain, heart, lungs, liver, spleen, kidneys, pancreas, small intestine, colon, adrenal glands, and bladder were removed and processed for pathological analysis. Tissues were evaluated by hematoxylin and eosin staining for evidence of toxicity with histoarchitectural changes by an independent blinded pathologist. This revealed no gross changes in histoarchitecture or immune infiltrates, consistent with a lack of toxicity. To establish whether JQAD1 is selective for degrading EP300 rather than CBP in vivo, we performed a 3D mouse model of Balb / c Crbn mice treated with vehicle control or JQAD1. ILE391VAL Immunohistochemistry for EP300 and CBP was performed on liver tissue from mice. This analysis demonstrated that JQAD1-treated animals had reduced EP300 protein expression levels in liver cell nuclei compared to vehicle-treated controls (Figure 12D). Consistent with the hypothesis that CBP may partially compensate for the loss of EP300, JQAD1-treated animals showed no histological or biochemical evidence of toxicity in the liver, and CBP remained detectable by immunohistochemistry despite the loss of EP300 in vivo (Figure 12D).

[0087] Table 1. JQAD1 had limited toxicity in vivo [Table 1]

[0088] EP300, but less commonly CBP, was identified as a dependency in neuroblastoma along with each of the members of MYCN and adrenergic CRC (Durbin et al. Nat. Genet. 50:1240-6 (2018)). Because EP300 catalyzes the H3K27ac mark, it was hypothesized that EP300 is preferentially involved in the high level expression of CRC master transcription factors. Because JQAD1 preferentially degraded EP300, a HAT that primarily catalyzes H3K27ac found in super-enhancers, it was reasoned that treatment with JQAD1 may have a major effect on the expression levels of genes in CRC. We therefore compared the effects of JQAD1 given daily for 14 days on the expression levels of several different classes of mRNAs, including those regulated by typical enhancers, super-enhancers, and all TFs as well as TFs encoding members of CRC (Figure 6E). This comparison revealed that the CRC gene, along with MYCN, was the most downregulated gene in JQAD1-treated tumors compared to other categories of genes. These results demonstrate that JQAD1 treatment dramatically downregulated the expression of a very important subset of genes encoding transcription factors, including CRC, including MYCN, the dominant oncogene in neuroblastoma cells and one on which neuroblastoma cells depend for proliferation and survival (Durbin et al. Nat. Genet. 50:1240-6 (2018); Pugh et al. Nat. Genet. 45:279-84 2013; Zeid et al. Nat Genet 50:515-23(2018)).

[0089] JQAD1 exhibited broad anti-neoplastic activity across cancer cell lines. Epigenetic and enhancer-mediated control of gene expression is necessary for normal cell and tissue developmental processes and is dysregulated in different cancer subtypes (reviewed in: Bradner et al. Cancer. Cell 168:629-43 (2017); Wimalasena et al. Mol Cell 78:1086-95 (2020)). In neuroblastoma cells, EP300 is the preferential regulator of H3K27ac, which exhibits active promoters and enhancers in addition to transcriptional activity. It was therefore hypothesized that there would be preferential dependence on EP300 or CBP across other cancer subtypes as well. The relative dependence of all available cell lines on EP300 or CBP was therefore tested using the DepMap genome-scale CRISPR-Cas9 loss-of-function screening dataset (Meyers et al. Nat. Genet. 49:1779-84 (2017)). Comparison of the probability of dependency on EP300 and CBP across a total of 757 human cancer cell lines representing 36 different tumor lineages demonstrated a higher probability of dependency on EP300 than on CBP across many cancer cell lines (p<0.0001, Figure 7A). Therefore, we stratified all cell lines in the DepMap by tumor lineage and examined the probability of dependency on EP300 and CBP in each lineage. This analysis showed that many tumor lines had an enhanced probability of dependency on EP300 compared to CBP (Figure 7B). A few tumor cell lines, particularly thyroid, pancreatic, and cervical cancers, showed an enhanced dependency on CBP compared to EP300 (Figure 7B).

[0090] Because the probability of dependency on EP300 was higher for many tumor lines than CBP, we evaluated whether JQAD1 would show antitumor effects across multiple tumor lines. We analyzed the response to JQAD1 in a pooled, barcoded, 5-day viability PRISM screen performed at the Broad Institute with 557 cancer cell lines (Figure 7C) (Corsello et al. Nat. Cancer 1:235-48 (2020)). These results demonstrate that JQAD1 treatment has antitumor activity across multiple tumor lines, many of which have enhanced dependency on EP300 compared to CBP. In most tumor lines, several exemplary cell lines showed growth inhibition with JQAD1 treatment (area under the curve (AUC) < 0.85) (Figure 7C). Because cell lines from multiple lineages showed growth inhibition with JQAD1 treatment, we evaluated whether predictors of JQAD1 activity could be determined. Therefore, we performed an analysis of the RNA expression profiles of all cell lines treated with JQAD1. Consistent with its mechanism of action, higher expression levels of CRBN correlated with higher JQAD1-mediated antineoplastic activity, as reflected by lower AUC measurements of the JQAD1 dose-response (Figure 7D), indicating that JQAD1 activity was at least partially determined by CRBN expression levels, consistent with the requirement by JQAD1 for CRBN to target EP300 for degradation.

[0091] To further explore this requirement, we hypothesized that increasing the expression level of CRBN in JQAD1-resistant cells could result in restoration of sensitivity. We therefore examined the response of BE2C neuroblastoma cells, which exhibit lower CRBN protein expression, to JQAD1 (Figure 10C). We established BE2C cells with stable overexpression of CRBN (BE2C-CRBN) or zsGreen (BE2C-zsGreen) as a control, and then treated the cells with either DMSO or JQAD1 (Figure 7E). EP300 was degraded within 24 h of treatment with JQAD1 in BE2C-CRBN cells, whereas the expression of EP300 in control cells was unaffected (Figure 7E). Consistent with these results, the proliferation of BE2C-CRBN cells was suppressed by JQAD1 treatment, whereas untreated BE2C-CRBN cells proliferated at a similar rate to BE2C-zsGreen cells treated with DMSO or JQAD1 (Figure 7F). Thus, overexpression of CRBN in JQAD1-insensitive BE2C cells was sufficient to restore sensitivity to the compound JQAD1. These data highlight that two important considerations for using degraders across different cell models include both the expression levels of key components of the PROTAC® machinery, such as CRBN, as well as individual cell line dependency on the PROTAC® target.

[0092] In summary, these data indicate that cancer cells, in addition to neuroblastoma, exhibit enhanced dependency on EP300 compared to CBP, and that JQAD1 is a potential way to exploit this enhanced dependency, particularly in individual tumors with elevated levels of CRBN expression.

[0093] The basis for the selective dependency on EP300, rather than CBP, in most pediatric neuroblastomas is demonstrated herein. In the adrenergic subtype of neuroblastoma, the AP2 family transcription factor TFAP2β has also been demonstrated to be a critical member of a core regulatory circuit that co-binds genome-wide with the remaining CRC factors. The core regulatory circuit is a lineage-defining autoregulatory transcription factor network that establishes the transcriptional landscape of distinct cell types (Boyer et al. Cell 122:947-56 (2005); Durbin et al. Nat. Genet. 50:1240-6 (2018); Saint-Andre et al. Genome Res. 26:385-96 (2016); Sanda et al. Cancer Cell 22:209-21 (2012); Wang et al. Nat. Commun. 10:5622 (2019)). Because EP300 and CBP do not recognize sequence-specific DNA motifs, they are transcription factor-dependent to localize to their target enhancers. Importantly, TFAP2β specifically binds to EP300, but not CBP, establishing the basis for dependency on EP300. Thus, TFAP2β specifically associates with EP300 at enhancers that form an extended regulatory network of adrenergic NB CRCs across the genome, including a network of genes that establish the malignant cell state in this subtype of neuroblastoma. Thus, loss of TFAP2β results in loss of H3K27ac marks on CRC-associated super-enhancers catalyzed by EP300 in neuroblastoma cells, thereby identifying TFAP2β as a dominant mediator of EP300 localization to critical super-enhancers. This mechanism results in direct regulation of lineage-specific and oncogenic loci in neuroblastomas through recruitment of EP300 by physical interaction with the novel CRC transcription factor TFAP2β. This function cannot be achieved by CBP, as it does not physically interact with TFAP2β, or indeed with other transcription factors in adrenergic CRC.In addition to transcription factors, other elements of the core regulatory circuitry, including enhancer RNAs and linker proteins such as LDB1 and LMO1, are essential components of this regulatory complex (Sanda et al. Cancer Cell 22:209-21 (2012); Suzuki et al. Cell 168:1000-14 (2017); Wang et al. Nat. Commun. 10:5622 (2019)). Evidence that coactivator proteins are found at genomic loci bound by CRC transcription factors and that loss of EP300 results in enhanced loss of CRC factor expression compared to other transcription factors in vivo led to the hypothesis that coactivator enzymes such as EP300 are important for the high level expression that defines genes in the CRC extended regulatory network, and that lineage- and tumor-specific CRC factors, such as TFAP2β in neuroblastoma, serve novel functions in the CRC complex that are required to recruit EP300 to establish a malignant cellular state (Sabari et al. Science 361:eaar3958 (2018)).

[0094] It has been demonstrated that activity through CRC targeted enhancers is required for cell proliferation and viability in adrenergic neuroblastoma (Durbin et al. Nat. Genet. 50:1240-6 (2018); Wang et al. Nat. Commun. 10:5622 (2019)). It is therefore not surprising that EP300 is a major dependency in neuroblastoma, whereas CBP is not in most NB cell lines, likely due to the lack of need to maintain high levels of expression of the network of genes driven by CRC in this disease.

[0095] In various cancer subtypes, the dependency on EP300 compared to CBP is significantly enhanced, highlighting the hypothesis that these two paralogous genes may play a context-dependent and distinct role in regulating cancer cell survival. As a result, selective targeting of EP300 in different types of cancer cell lines that depend on EP300 may be effective in eliciting antitumor activity with reduced toxicity, since CBP may still be active in normal cells and may be able to compensate for the loss of EP300 in most normal cells. This attractive hypothesis has been difficult to test due to the significant homology between these two proteins, which has prevented pharmacological strategies that preferentially target one of these enzymes while leaving the other active.

[0096] The PROTAC® JQAD1 is selective in its ability to degrade EP300 compared to CBP, dependent on the binding activity of A485, as described herein. This observation contrasts sharply with the more promiscuous acetyltransferase inhibitory activity of A485 against both EP300 and CBP. PROTAC® agents synthesized from bait molecules that bind to several closely related proteins sometimes exhibit substrate specificity, such as with bromodomain-containing protein 4 (BRD4) and p38 degraders (reviewed in Burslem and Crews, Cell 181:102-14 (2020)). The mechanism of this selectivity is thought to be related to three-dimensional interactions between the chimeric degrader compound and the E3 ligase complex, mediated by the three-dimensional structure of the target protein and the E3 ligase receptor. Due to the size and lack of solubility of the full-length EP300 and CBP proteins, full-length crystal structures have not been solved. However, biotin-JQAD1 forms a ternary complex with EP300 and CRBN, without CBP. Thus, in contrast to A485, which has comparable activity towards EP300 and CBP, JQAD1 bound more strongly to EP300 in biochemical assays.

[0097] JQAD1 has several interesting properties: i) it showed selectivity for EP300 compared to CBP in multiple neuroblastoma cell types; ii) it had higher potency than the parent inhibitor in several cell types; and iii) it was useful in degrading EP300 with limited effect on CBP and limited toxicity in vivo. EP300 was degraded by JQAD1 in vivo in normal mouse tissues expressing humanized CRBN, but CBP staining was only minimally affected in these tissues. Furthermore, these tissues showed normal architecture. These data support the hypothesis that CBP compensates for the loss of EP300 in some normal tissues. Thus, after profiling blood counts, liver and kidney function tests, body weight, and grooming, mice treated with 40mg / Kg JQAD1 IP twice daily for 14 days were observed with no toxicity. Thus, it is hypothesized that CBP-mediated activity can at least partially compensate for the loss of EP300 in non-transformed cells.

[0098] Experiments using JQAD1 also allowed the identification of a biased activity towards loss of H3K27ac signal, prior to the effects of expression of genes forming an extended regulatory network in CRC. JQAD1 caused selective degradation of full-length EP300, compared to catalytic inhibition of EP300 and CBP by A485. This indicates that loss of full-length EP300 causes induction of apoptosis in neuroblastoma cells, compared to catalytic inhibition. In neuroblastoma, EP300 physically interacts with the dominant tumor oncoprotein MYCN and controls its localization to chromatin. Thus, degradation of EP300 leads to loss of this binding activity, which then leads to dissociation of MYCN from chromatin. Previous evidence indicates that MYCN, and indeed other MYC proteins, broadly engage chromatin to drive enhancer invasion and are independently required to suppress apoptosis in neuroblastoma cells (Huang and Weiss, Cold Spring Harb Perspect Med. 3:a014415 (2013); Zeid et al. Nat Genet 50, 515-23 (2018)). Thus, these data suggest a novel mechanism by which MYCN is maintained in a chromatin-bound state via physical interaction with EP300, thereby facilitating MYCN-mediated enhancement of enhancer invasion and CRC-based oncogenic transcription.

[0099] Thus, distinct roles of EP300 and CBP in regulating cell proliferation in high-risk pediatric neuroblastoma are described herein. These findings are similarly identified in various other tumor types, indicating that enhanced dependency on EP300 is a common finding in human cancer. EP300, but not CBP, is required for the regulation of H3K27ac and the gene expression landscape of a subset of high-risk neuroblastomas. This function is performed by the interaction between EP300 and the novel CRC transcription factor TFAP2β, which mediates the binding of EP300 to enhancers and promoters associated with CRC. In doing so, TFAP2β and EP300 jointly determine gene expression patterns in the adrenergic subtype of high-risk neuroblastoma. The PROTAC® JQAD1 was generated to take advantage of these findings. Importantly, loss of EP300 results in the dissociation of the dominant neuroblastoma oncoprotein MYCN from chromatin, resulting in loss of enhancer invasion, suppression of CRC-based transcription and apoptosis. These data provide important insights into enhancer regulation in high-risk neuroblastoma and highlight a new paradigm for chemical epigenetic control of gene enhancer and mRNA expression in high-risk neuroblastoma, with relevance to other types of human cancers.

[0100] World Health Organization standards. The WHO criteria for assessing the efficacy of anticancer drugs on tumor shrinkage, developed in the 1970s by the International Union Against Cancer and the World Health Organization, represent the first generally agreed upon specific criteria for the systematization of tumor response assessment. These criteria were first published in 1981 (Miller et al. 1981 Clin. Cancer Res., 47:207-14). The WHO criteria proposed >50% tumor shrinkage for partial response and >25% tumor increase for progressive disease.

[0101] Response Evaluation Criteria in Solid Tumors (RECIST). RECIST is a set of published rules that define when tumors in cancer patients improve ("respond"), stay the same ("stable"), or get worse ("progress") during treatment (Eisenhauer et al. 2009 European Journal of Cancer, 45:228-247). Only patients with measurable disease at baseline should be included in protocols where objective tumor response is the primary endpoint. Response criteria for evaluation of target lesions were as follows: Complete response (CR): Disappearance of all target lesions. Partial Response (PR): At least a 30% reduction in the sum of the longest diameters (LD) of target lesions, based on baseline sum LD. · Stable Disease (SD): Based on the minimum total LD ​​from the start of treatment, there is neither sufficient shrinkage to be considered PR nor sufficient increase to be considered PD. Progressive disease (PD): at least a 20% increase in the sum of the LD of target lesions or the appearance of one or more new lesions, based on the smallest total LD ​​recorded since treatment was started. Response criteria for evaluation of non-target lesions were as follows: Complete response (CR): Disappearance of all non-target lesions and normalization of tumor marker levels. · Incomplete response / Stable disease (SD): persistence of one or more non-target lesions and / or maintenance of tumor marker levels above normal limits. Progressive Disease (PD): Appearance of one or more new lesions and / or overt progression of existing non-target lesions.

[0102] The response criteria for the evaluation of best overall response are as follows: Best overall response is the best response recorded from the start of treatment to disease progression / relapse (the criterion for PD is the minimum measurement recorded since treatment was started). In general, a patient's best response assignment depends on the achievement of both measurement and confirmation criteria. Patients with an overall deterioration in health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be classified as having "symptomatic deterioration." Every effort should be made to document objective progression even after cessation of treatment. In some situations, it may be difficult to distinguish residual disease from normal tissue. Where assessment of complete response relies on this determination, it is recommended to investigate residual disease (fine needle aspiration / biopsy) to confirm complete response status.

[0103] Immune-Related Response Criteria The immune-related response criteria (irRC) are a set of published rules that define when tumors in cancer patients improve ("respond"), stay the same ("stable"), or worsen ("progress") during treatment, and the compounds being evaluated are immuno-oncology drugs. First published in 2009 (Wolchok et al. Clin. Cancer Res. 15:7412 (2009)), the immune-related response criteria arose from the observation that in clinical trials that measured response using WHO or RECIST criteria, immuno-oncology drugs failed because these criteria failed to account for the time gap between initial treatment and the apparent action of the immune system to reduce tumor burden in many patients. An important impetus in the development of irRC was the observation that in studies of various cancer therapies derived from the immune system, such as cytokines and monoclonal antibodies, the expected complete and partial responses as well as stable disease occurred only after an increase in tumor burden that the traditional RECIST criteria would translate to "progressive disease." Because RECIST fails to take into account the delay between dosing and the observed antitumor T-cell response, otherwise "successful" drugs, i.e., drugs that ultimately extended life, failed in clinical trials.

[0104] The irRC is based on WHO criteria, but measurements of tumor burden and assessment of immune-related responses have been modified as described below.

[0105] Measurement of tumor burden In irRC, tumor burden is measured by combining "index" lesions with new lesions. Typically, tumor burden is measured using a limited number of "index" lesions (i.e., the largest identifiable lesions) at baseline, and new lesions identified at subsequent time points are counted as "progressive disease." In irRC, in contrast, new lesions are a change in tumor burden. irRC retained the bidirectional measurement of lesions originally stipulated in the WHO criteria.

[0106] Assessment of immune-related responses In irRC, an immune-related complete response (irCR) is the disappearance of all lesions (measured or not) and no new lesions; an immune-related partial response (irPR) is a 50% decrease in tumor burden from baseline as defined by irRC; an immune-related progressive disease (irPD) is a 25% increase in tumor burden from the lowest level recorded. All others are considered immune-related stable disease (irSD). Even if the tumor burden is rising, the immune system is likely to "work" several months after the first dose, resulting in an eventual decrease in tumor burden for many patients. The 25% threshold accounts for this apparent delay.

[0107] Gene expression profiling In general, methods of gene expression profiling can be divided into two major groups: methods based on polynucleotide hybridization analysis and methods based on polynucleotide sequencing. Methods known in the art for quantification of mRNA expression in a sample include Northern blotting and in situ hybridization, RNAse protection assay, RNA-seq, and reverse transcription polymerase chain reaction (RT-PCR). Alternatively, antibodies that recognize specific duplexes, such as DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes, are used. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS). For example, RT-PCR is used to compare mRNA levels in different sample populations, normal tissues, and tumor tissues with or without drug treatment, to characterize patterns of gene expression (i.e., expression levels), to distinguish closely related mRNAs, and / or to analyze RNA structure.

[0108] In some cases, the first step in gene expression profiling by RT-PCR is reverse transcription of RNA template into cDNA, followed by amplification in a PCR reaction.For example, extracted RNA is reverse transcribed using GeneAmp RNA PCR kit (Perkin Elmer, Calif., USA) according to the manufacturer's instructions.The cDNA is then used as template in subsequent PCR amplification and quantitative analysis, for example, using TaqMan™ Respiratory Tract Microbiota (RTM) (Life Technologies™, Inc., Grand Island, NY) assay.

[0109] Microarray. Differential gene expression can also be identified or confirmed using microarray technology. In these methods, polynucleotide sequences of interest (including cDNA and oligonucleotides) are plated or arrayed on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. As with the RT-PCR method, the source of mRNA is typically total RNA isolated from human tumors or tumor cell lines and corresponding normal tissues or cell lines. Thus, RNA is isolated from various primary tumors or tumor cell lines. When the source of mRNA is a primary tumor, mRNA is extracted from frozen or archived tissue samples.

[0110] In microarray technology, PCR-amplified inserts of cDNA clones are applied to a substrate in a high-density array. The microarrayed genes immobilized on the microchip are suitable for hybridization under stringent conditions.

[0111] In some cases, fluorescently labeled cDNA probes are generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest (e.g., leukemia tissues). Labeled cDNA probes applied to the chip specifically hybridize to DNA loci on the array. After washing to remove non-specifically bound probes, the chip is scanned by a confocal laser microscope or another detection method such as a charge-coupled device (CCD) camera. Quantification of hybridization of each array element allows assessment of the corresponding mRNA abundance.

[0112] In some configurations, dual-color fluorescence is used. With dual-color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. Thus, the relative abundance of the transcripts from the two sources corresponding to each specific gene is determined simultaneously. In various configurations, miniaturized scale hybridization can provide a simple and rapid evaluation of the expression pattern for a large number of genes. In various configurations, such methods can have the necessary sensitivity to detect rare transcripts that are expressed at less than 1000, 100, or 10 copies per cell. In various configurations, such methods can detect at least approximately two-fold differences in expression levels (Schena et al. Proc. Natl. Acad. Sci. USA 93:106-149 (1996)). In various configurations, microarray analysis is performed by commercially available equipment according to manufacturer's protocols, such as by using Affymetrix GenChip technology or Incyte's microarray technology.

[0113] RNA sequencing (RNA-seq), also known as whole transcriptome shotgun sequencing (WTSS), is another technique for identifying or confirming differential gene expression. RNA-seq uses next-generation sequencing (NGS) to reveal the presence and amount of RNA in a biological sample at a given time point.

[0114] RNA-Seq is used to analyze the continuously changing cellular transcriptome. See, e.g., Wang et al. Nat. Rev. Genet. 10:57-63 (2009). Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced ​​transcripts, post-transcriptional modifications, gene fusions, mutations / SNPs, and changes in gene expression. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA including total RNA, miRNA, small RNAs such as tRNA, and ribosome profiling. RNA-Seq can also be used to determine exon / intron boundaries and validate or revise previously annotated 5' and 3' gene boundaries.

[0115] Before RNA-Seq, gene expression studies were performed using hybridization-based microarrays. Problems with microarrays include cross-hybridization artifacts, poor quantification of low- and high-expressing genes, and the need to know the sequence of interest. Due to these technical issues, transcriptomics moved to sequencing-based methods. These progressed from Sanger sequencing of Expressed Sequence Tag libraries, to chemical tag-based methods (e.g., serial analysis of gene expression), and finally to the current technology of NGS of cDNA (specifically, RNA-Seq).

[0116] Pharmaceutical Treatment Agents For therapeutic use, the agents described herein (e.g., JQAD1) can be formulated in a pharma- ceutically acceptable buffer, such as saline, and administered systemically. Preferred routes of administration include, for example, subcutaneous, intravenous, intraperitoneal, intramuscular, or intradermal injections, which provide a continuous, sustained level of drug in the patient. Treatment of human patients or other animals is performed using a therapeutically effective amount of the therapeutic agent identified herein in a physiologically acceptable carrier. Suitable carriers and their formulations are described, for example, in Remington's Pharmaceutical Sciences by EW Martin. The amount of agent administered will vary depending on the mode of administration, the age and weight of the patient, and the clinical symptoms of (e.g., NB). In general, dosages will be within the range of amounts used for other agents used in the treatment of other diseases associated with EP300 dependency (e.g., cancer (e.g., NB)), although in some cases, lesser amounts may be required due to increased specificity of the agent. For example, the agent is administered at a dosage that is cytotoxic to neoplastic cells.

[0117] In one aspect, the disease or disorder is cancer.In certain embodiments, the cancer is solid tumor, such as neuroblastoma, rhabdomyosarcoma, malignant melanoma, colon cancer, rectal cancer, gastric cancer, breast cancer, brain cancer and pancreatic cancer.In certain embodiments, the cancer is blood cancer, such as leukemia, myeloma and lymphoma.In certain embodiments, the cancer is high-risk neuroblastoma.In some embodiments, the EP300-dependent cancer is high-risk NB.

[0118] formulation As those skilled in the art will recognize that modifying dosages for humans compared to animal models is routine in the art, human dosages can be determined initially by extrapolating from the amount of drug used in animal models.In certain embodiments, it is envisioned that dosages can vary between about 1 μg compound / Kg body weight to about 5000 mg compound / Kg body weight; or about 5 mg / Kg body weight to about 4000 mg / Kg body weight or about 10 mg / Kg body weight to about 3000 mg / Kg body weight; or about 50 mg / Kg body weight to about 2000 mg / Kg body weight; or about 100 mg / Kg body weight to about 1000 mg / Kg body weight; or about 150 mg / Kg body weight to about 500 mg / Kg body weight. In other cases, the dose may be about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 mg / Kg body weight. In other embodiments, it is contemplated that the dose may range from about 5 mg compound / Kg body weight to about 20 mg compound / Kg body weight. In other embodiments, the dose may be about 8, 10, 12, 14, 16 or 18 mg / Kg body weight. Of course, this dosage may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of initial clinical trials and the needs of the particular patient.

[0119] In some cases, the agents of the invention are administered at doses lower than the human equivalent dosage (HED) of the no observed adverse effect level (NOAEL) for a period of 3 months, 4 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years or more. The NOAEL determined in animal studies is useful for determining the maximum recommended starting dose for human clinical trials. For example, the NOAEL can be extrapolated to determine a human equivalent dosage. Typically, such extrapolations between species are performed using doses normalized to body surface area (i.e., mg / m2). 2 In certain embodiments, the NOAEL is determined in mice, hamsters, rats, ferrets, guinea pigs, rabbits, dogs, primates, primates (monkeys, marmosets, squirrel monkeys, baboons), micropigs or minipigs. For a discussion of the use of NOAELs to determine human equivalent doses and their extrapolation, see Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, US Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER), Pharmacology and Toxicology, July 2005.

[0120] The amount of the agent of the present invention used in the preventive and / or therapeutic regimen that is effective in the treatment of hematopoietic cancer or autoimmune disease can be based on the currently prescribed dosage of the agent, and can be evaluated by the methods disclosed herein and known in the art. The frequency and dosage also vary according to factors specific to each subject, depending on the specific agent administered, the severity of the cancerous condition, the route of administration, and the age, weight, response, and stab of the subject. For example, the dosage of the agent of the present invention that is effective in the treatment of cancer can be determined by administering the agent to an animal model, such as the animal model disclosed herein or known to those skilled in the art. In addition, in vitro assays can be used as necessary to help identify optimal dosage ranges.

[0121] In some embodiments, the prophylactic and / or therapeutic regimens include titrating the dosage administered to the patient to achieve a particular measure of therapeutic efficacy, including a reduction in the cancer cell population in the patient.

[0122] In some cases, the dosage of the agent of the present invention in the prophylactic and / or therapeutic regimen is adjusted to achieve a reduction in the number or amount of cancer cells found in the test specimen extracted from the patient after undergoing the prophylactic and / or therapeutic regimen, compared with the reference sample. Here, the reference sample is a specimen extracted from the patient undergoing the treatment, and the specimen is extracted from the patient at an earlier time point. In one embodiment, the reference sample is a specimen extracted from the same patient before undergoing the prophylactic and / or therapeutic regimen. For example, the number or amount of cancer cells in the test specimen is at least 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% lower than in the reference sample.

[0123] In some cases, the dosage of the agent of the present invention in a prophylactic and / or therapeutic regimen is adjusted to achieve a number or amount of cancer cells that falls within a predetermined reference range. In these embodiments, the number or amount of cancer cells in a test specimen is compared to a predetermined reference range.

[0124] In other embodiments, the dosage of the agent of the present invention in the prophylactic and / or therapeutic regimen is adjusted to achieve a reduction in the number or amount of cancer cells found in the test sample extracted from the patient after undergoing the prophylactic and / or therapeutic regimen, compared to a reference sample, the reference sample being a sample extracted from a healthy non-cancer patient.For example, the number or amount of cancer cells in the test specimen is within at least 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% of the number or amount of cancer cells in the reference sample.

[0125] In treating certain human patients with solid tumors, extracting multiple tissue specimens from suspected tumor sites may prove impractical. In these cases, the dosage of the agent of the present invention in a prophylactic and / or therapeutic regimen for human patients is extrapolated from the dose in the animal model that is effective in reducing the cancer population in the animal model. In the animal model, the prophylactic and / or therapeutic regimen is adjusted to achieve a reduction in the number or amount of cancer cells found in a test specimen extracted from the animal after undergoing the prophylactic and / or therapeutic regimen compared to a reference sample. The reference sample can be a specimen extracted from the same animal before undergoing the prophylactic and / or therapeutic regimen. In a specific embodiment, the number or amount of cancer cells in the test specimen is at least 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50% or 60% less than in the reference sample. The dose effective in reducing the number or amount of cancer cells in an animal is determined based on the body surface area (e.g., mg / m2). 2 ) to provide an equivalent human dose.

[0126] The prophylactic and / or therapeutic regimens disclosed herein include administration of an agent of the present invention or a pharmaceutical composition thereof to a patient in a single dose or in multiple doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more doses).

[0127] In one embodiment, the prophylactic and / or therapeutic regimen comprises administering the agent of the present invention or its pharmaceutical composition in multiple doses.When administered in multiple doses, the agent or pharmaceutical composition is administered with sufficient frequency and amount to treat the condition.For example, the administration frequency ranges from once a day to about once every 8 weeks.In another example, the administration frequency ranges from about once a week to about once every 6 weeks.In another example, the administration frequency ranges from about once every 3 weeks to about once every 4 weeks.

[0128] In general, the dosage of the agent of the present invention administered to a subject for treating cancer is in the range of 0.01 to 500 mg / Kg of the subject's body weight, for example, in the range of 0.1 mg / Kg to 100 mg / Kg. For example, the dosage administered to a subject is in the range of 0.1 mg / Kg to 50 mg / Kg or 1 mg / Kg to 50 mg / Kg of the subject's body weight, more preferably in the range of 0.1 mg / Kg to 25 mg / Kg or 1 mg / Kg to 25 mg / Kg of the patient's body weight. In another example, the dosage of an agent of the present invention administered to a subject to treat cancer in the patient is 500mg / Kg or less, preferably 250mg / Kg or less, 100mg / Kg or less, 95mg / Kg or less, 90mg / Kg or less, 85mg / Kg or less, 80mg / Kg or less, 75mg / Kg or less, 70mg / Kg or less, 65mg / Kg or less, 60mg / Kg or less, 55mg / Kg or less, 50mg / Kg or less, 45mg / Kg or less, 40mg / Kg or less, 35mg / Kg or less, 30mg / Kg or less, 25mg / Kg or less, 20mg / Kg or less, 15mg / Kg or less, 10mg / Kg or less, 5mg / Kg or less, 2.5mg / Kg or less, 2mg / Kg or less, 1.5mg / Kg or less, or 1mg / Kg or less of the patient's body weight.

[0129] In another example, the dosage of the agent of the present invention administered to a subject to treat cancer in the patient is a unit dose of 0.1 to 50 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[0130] In another example, the dose of the agent of the invention administered to a subject to treat the patient's cancer is between 0.01 and 10 g / m of the subject's body weight. 2 in the range of 0.1 g / m 2 ~7.5g / m 2 For example, the dose administered to a subject is in the range of 0.5 g / m2 of the subject's body surface area. 2 ~5g / m 2 , or 1 g / m 2 ~5g / m 2 The range is.

[0131] In another example, the prophylactic and / or therapeutic regimen comprises administering to the patient one or more doses of an effective amount of an agent of the invention, wherein the effective amount of the dose achieves a plasma level of the agent of the invention of at least 0.1 μg / mL, at least 0.5 μg / mL, at least 1 μg / mL, at least 2 μg / mL, at least 5 μg / mL, at least 6 μg / mL, at least 10 μg / mL, at least 15 μg / mL, at least 20 μg / mL, at least 25 μg / mL, at least 50 μg / mL, at least 100 μg / mL, at least 125 μg / mL, at least 150 μg / mL, at least 175 μg / mL, at least 200 μg / mL, at least 225 μg / mL, at least 250 μg / mL, at least 275 μg / mL, at least 300 μg / mL, at least 325 μg / mL, at least 350 μg / mL, at least 375 μg / mL, or at least 400 μg / mL.

[0132] In another example, the prophylactic and / or therapeutic regimen comprises administering to the patient multiple doses of an effective amount of the agent of the invention, the multiple doses being at least 0.1 μg / mL, at least 0.5 μg / mL, at least 1 μg / mL, at least 2 μg / mL, at least 5 μg / mL, at least 6 μg / mL, at least 10 μg / mL, at least 15 μg / mL, at least 20 μg / mL, at least 25 μg / mL, at least 50 μg / mL, at least 100 μg / mL, at least 125 μg / mL, at least 150 μg / mL , at least 175 μg / mL, at least 200 μg / mL, at least 225 μg / mL, at least 250 μg / mL, at least 275 μg / mL, at least 300 μg / mL, at least 325 μg / mL, at least 350 μg / mL, at least 375 μg / mL, or at least 400 μg / mL, for at least 1 day, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, or 36 months.

[0133] Combination therapy In one example, the drug is administered in combination therapy, i.e., combined with other drugs, such as therapeutic agents, that are useful for treating pathological conditions or disorders, such as various forms of cancer.The term "combined" in this context means that the drug is administered substantially contemporaneously, simultaneously or sequentially.When administered sequentially, at the start of administration of the second compound, the first compound of the two compounds can still be detected at effective concentrations at the site of treatment, if necessary.

[0134] Administration of the compound or combination of compounds for the treatment of neoplasms may be by any suitable means that results in a concentration of therapeutic agent that, in combination with other ingredients, is effective to ameliorate, reduce or stabilize the neoplasm. The agent may be contained in any suitable amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The agent may be provided in a dosage form suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal) administration route. The agent may be formulated according to conventional pharmaceutical practice (see, for example, Remington: The Science and Practice of Pharmacy (20th ed.), ed. AR Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York).

[0135] Thus, in some examples, the prophylactic and / or therapeutic regimen comprises the administration of the agent of the present invention in combination with one or more additional anticancer therapeutics.In one example, the dosage of one or more additional anticancer therapeutics used in combination therapy is lower than the dosage that has been or is currently used to treat cancer.The recommended dosage of one or more additional anticancer therapeutics currently used to treat cancer can be obtained from any reference in the field, including but not limited to Hardman et al. eds., Goodman & Gilman's The Pharmacological Basis of Basis of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; Physician's Desk Reference (60.sup.th ed., 2006).

[0136] In some embodiments, the agents of the invention may be used in combination with one or more additional anti-cancer therapeutic agents. Examples of anti-cancer therapeutic agents include cis-retinoic acid, cyclophosphamide (Cytoxan®, Neosar®, Endoxan®), cisplatin (Platinol®), carboplatin (Paraplatin®), vincristine (Oncovin®, Vincasar PFS®, VCR), doxorubicin (Adriamycin®, Rubex®), etoposide (Toposar®, VePesid®, Etopophos®, VP-16), topotecan (Hycamtin®), busulfan (Myleran®, Busulfex®), and melphalan (Alkeran®, L-PAM, Evomela®), or thiotepa (Thioplex®, Tepadina®).

[0137] In some embodiments, the anti-cancer therapeutic comprises methylprednisolone (Depo-Medrol®, Solu-Medrol®, Medrol®), prednisone (Sterapred®, Prednisone Intensol), dexamethasone (Decadron®), hydrocortisone (Cortef®), or adrenocorticotropic hormone derivatives including tetracosactide (synacthen®, tetracosactrin®, cosyntropin®).

[0138] In some embodiments, the prophylactic and / or therapeutic regimen comprises administration of an agent of the invention in combination with a combination chemotherapeutic agent. In some embodiments, the combination chemotherapy agent is busulfan (Myleran®, Busulfex®), carboplatin (Paraplatin®) or cisplatin (Platinol®), cyclophosphamide (Cytoxan®, Neosar®, Endoxan®), doxorubicin (Adriamycin®, Rubex®), etoposide (Toposar®, VePesid®, Etopophos®, VP-16), irinotecan (Onivyde®), temozolomide (Temodal®, or ifosfamide (Ifex®), thiotepa (Tepadina®), melphalan (Evomela®), topotecan (Hycamtin®), or vincristine (Marqibo®, Vincasar®). PFS®). In some embodiments, this treatment is followed by stem cell transplantation. Chemotherapy agents can be used in monotherapy (i.e., a single chemotherapy agent) or in combination with other treatments as polytherapy (i.e., multiple chemotherapy agents). Polytherapy can include any combination of agents. One common polytherapy includes cisplatin (or carboplatin), cyclophosphamide, doxorubicin, vincristine, and etoposide.

[0139] In some cases, prophylactic and / or therapeutic regimens include administration of an agent of the present invention in combination with an immunosuppressant, such as dinutuximab (Unituxin®), with or without cis-retinoic acid, or rituximab (Rituxan®).

[0140] The agents of the invention and the one or more additional anti-cancer therapeutic agents may be administered separately, simultaneously, or sequentially. In various embodiments, the agents of the invention and the additional anticancer therapeutic agent are administered less than 5 minutes apart, less than 30 minutes apart, less than 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 to about 3 hours apart, about 3 to about 4 hours apart, about 4 to about 5 hours apart, about 5 to about 6 hours apart, about 6 to about 7 hours apart, about 7 to about 8 hours apart, about 8 to about 9 hours apart, about 9 to about 10 hours apart, about 10 to about 11 hours apart, about 11 to about 12 hours apart, about 12 to 18 hours apart, 18 to 24 hours apart, 24 to 36 hours apart, 36 to 48 hours apart, 48 to 52 hours apart, 52 to 60 hours apart, 60 to 72 hours apart, 72 to 84 hours apart, 84 to 96 hours apart, 96 hours apart, 120 hours apart, or 168 hours apart. In another example, two or more anti-cancer therapeutic agents are administered within the same patient visit.

[0141] In certain embodiments, the agent of the present invention and the additional anticancer therapeutic agent are administered periodically. Cycling therapy includes administering one anticancer therapeutic agent for a period of time, then administering a second anticancer therapeutic agent for a period of time, and repeating this sequential administration, i.e., cycle, to reduce the occurrence of resistance to one or both of the agents, to avoid or reduce the side effects of one or both of the agents, and / or to improve the effectiveness of the therapy. In one example, cycling therapy includes administering a first anticancer therapeutic agent for a period of time, then administering a second anticancer therapeutic agent for a period of time, and optionally administering a third anticancer therapeutic agent for a period of time, etc., and repeating this sequential administration, i.e., cycle, to reduce the occurrence of resistance to the agents, to avoid or reduce the side effects of one of the agents, and / or to improve the effectiveness of the agents.

[0142] In another example, the agents are administered simultaneously to a subject in separate compositions.Agent combinations of the present invention can be administered to a subject by the same or different routes of administration.

[0143] When the agent of the present invention and the additional anti-cancer therapeutic agent are administered to a subject at the same time, the term "simultaneously" does not mean to be limited to administration of the agents at exactly the same time, but rather to be administered to a subject in an order and time interval such that they can act together (e.g., act synergistically to provide increased benefits than if they were administered otherwise). For example, the agents can be administered at the same time or at different times sequentially in any order, but if not administered at the same time, they should be administered close enough in time to provide the desired therapeutic effect, preferably in a synergistic manner. The combination of agents can be administered separately in any suitable form and by any suitable route. It is understood that if the components of the combination of agents are not administered in the same pharmaceutical composition, they can be administered to a subject in need thereof in any order. For example, an agent of the invention can be administered to a subject in need thereof prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional anti-cancer therapeutic agent. In various embodiments, the agents are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1-2 hours apart, 2-3 hours apart, 3-4 hours apart, 4-5 hours apart, 5-6 hours apart, 6-7 hours apart, 7-8 hours apart, 8-9 hours apart, 9-10 hours apart, 10-11 hours apart, 11-12 hours apart, 24 hours or less apart, or 48 hours or less apart. In one example, the agents are administered within the same clinic visit. In another example, a combination of agents of the present invention is administered 1 minute to 24 hours apart.

[0144] Release of pharmaceutical composition The pharmaceutical compositions according to the invention can be formulated to release the drug substantially immediately after administration or at any predetermined time or period after administration. The latter type of composition is generally known as a controlled release formulation, and includes: (i) formulations that produce a substantially constant concentration of drug in the body over an extended period of time; (ii) formulations that produce a substantially constant concentration of drug in the body over an extended period of time after a predetermined lag time; (iii) formulations that sustain action during a predetermined period of time by maintaining a relatively constant effective level in the body while simultaneously minimizing undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action, for example, by spatial location of the controlled release composition adjacent to or in contact with the thymus; (v) formulations that allow convenient dosing, for example, a dose administered once a week or once every two weeks; and (vi) formulations that target neoplasms by using carriers or chemical derivatives to deliver the drug to a specific cell type (e.g., neoplastic cells). In some applications, controlled release formulations eliminate the need for frequent administration during the day to maintain plasma levels at therapeutic levels.

[0145] In order to obtain controlled release, in which the release rate exceeds the metabolism rate of the drug, any of many strategies can be implemented.In one example, controlled release can be obtained by appropriate selection of various formulation parameters and ingredients, including, for example, various types of controlled release compositions and coatings.Therefore, the therapeutic agent is formulated with suitable excipients into a pharmaceutical composition that releases the therapeutic agent in a controlled manner when administered.Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

[0146] Parenteral Compositions Pharmaceutical compositions can be administered parenterally through injection, infusion, or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) in dosage forms, formulations, or suitable delivery devices or implants that contain conventional non-toxic pharmaceutically acceptable carriers and adjuvants.The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation.Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

[0147] Compositions for parenteral use may be provided in unit dosage form (e.g., single-dose ampoules) or in vials containing several doses and to which suitable preservatives may be added (see below). The compositions may be in the form of a solution, suspension, emulsion, injection device, or implantable delivery device, or may be provided as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the agent that reduces or improves neoplasms, the composition may include suitable parenterally acceptable carriers and / or excipients. The agent may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, etc. for controlled release. In addition, the composition may include suspending agents, solubilizing agents, stabilizing agents, pH agents, tonicity adjusting agents, and / or dispersing agents.

[0148] As mentioned above, the pharmaceutical compositions according to the invention may be in a form suitable for sterile injection. To prepare such compositions, the appropriate active antineoplastic therapeutic agent is dissolved or suspended in a parenterally acceptable liquid vehicle. Among the acceptable vehicles and solvents that may be used are water, water adjusted to a suitable pH by the addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. Aqueous formulations may also contain one or more preservatives (e.g., methyl, ethyl, or n-propyl p-hydroxybenzoate). If one of the compounds is poorly soluble or sparingly soluble in water, a dissolution enhancer or solubilizer can be added, or the solvent can include 10-60% w / w propylene glycol.

[0149] Controlled Release The controlled release parenteral compositions may be in the form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions. Alternatively, the active drug may be incorporated into biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.

[0150] Materials for use in preparing microspheres and / or microcapsules are biodegradable / biodegradable polymers, such as polygalactin, poly-(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamine) and poly(lactic acid). Biocompatible carriers that can be used when formulating controlled release parenteral formulations are carbohydrates (e.g., dextran), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be non-biodegradable (e.g., polydimethylsiloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid), or poly(orthoester), or combinations thereof).

[0151] Pharmaceutical Kits The composition of the present invention can be assembled into a pharmaceutical kit for use in improving neoplasms.The pharmaceutical kit according to this aspect of the present invention comprises carrier means, such as a box, carton, tube, etc., having one or more container means, such as a vial, tube, ampoule, or bottle, in close confinement therein.The pharmaceutical kit of the present invention can also comprise associated instructions for using the agent of the present invention.

[0152] How to use In some aspects, the present invention is directed to a method of treating a disease or disorder associated with abnormal (e.g., dysfunctional or dysregulated) EP300 activity, referred to herein as an "EP300-dependent" disease or disorder, wherein the treatment involves administering to a subject in need thereof a therapeutically effective amount of a selective degrader of EP300 (e.g., JQAD1) or a pharma- ceutical acceptable salt or stereoisomer thereof.

[0153] These EP300-dependent diseases or disorders are characterized by abnormal EP300 activity (e.g., elevated levels of EP300 or otherwise functionally abnormal EP300 compared to non-pathological conditions). A "disease" is generally considered to be a condition in which a subject is unable to maintain homeostasis and the subject's health condition will continue to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in a subject is a health condition in which the subject is able to maintain homeostasis, but the subject's health condition is less favorable than if the disorder were not present. If left untreated, the disorder does not necessarily cause further deterioration of the animal's health condition. In some embodiments, the compounds of the present application may be useful in the treatment of cell proliferation diseases and disorders (e.g., cancer or benign neoplasms). As used herein, the term "cell proliferation disease or disorder" refers to a condition characterized by deregulated or abnormal cell proliferation, or both, including neoplasms, precancerous conditions, benign tumors, and noncancerous conditions such as cancer.

[0154] The term "subject" (or "patient") as used herein includes all members of the animal kingdom susceptible to or afflicted with the indicated disease or disorder. In some embodiments, the subject is a mammal, e.g., a human or non-human mammal. The method is also applicable to companion animals such as dogs and cats, as well as livestock such as cows, horses, sheep, goats, pigs, and other domesticated and wild animals. A subject "in need" of treatment according to the present invention may be a subject "suffering or suspected of suffering from" a particular disease or disorder, may have been positively diagnosed, or may otherwise exhibit a sufficient number of risk factors or a sufficient number or combination of signs or symptoms to allow a medical professional to diagnose or suspect the subject as suffering from the disease or disorder. Thus, subjects suffering from and suspected of suffering from a particular disease or disorder are not necessarily two distinct groups.

[0155] The term "sample" as used herein refers to a biological sample obtained for the purpose of in vitro evaluation. Exemplary tissue samples for the methods described herein include tissue samples from NB tumors or the surrounding tumor microenvironment (i.e., stroma). The tumor microenvironment, typically composed of proliferating tumor cells, tumor stroma, blood vessels, infiltrating inflammatory cells, and various associated tissue cells, is unique and emerges throughout the course of tumor progression as a result of its interactions with the host. It is created and governed by the tumor, and it influences and drives the molecular and cellular events that occur in the surrounding tissues. For the methods disclosed herein, the sample or patient sample may preferably include any bodily fluid or tissue. In some embodiments, bodily fluids include, but are not limited to, blood, plasma, serum, lymph, breast milk, saliva, mucus, semen, vaginal secretions, cell extracts, inflammatory fluids, cerebrospinal fluid, feces, vitreous humor, or urine obtained from a subject. In some aspects, the sample is a composite panel of at least two of a blood sample, a plasma sample, a serum sample, and a urine sample. In exemplary embodiments, the sample comprises blood or a fraction thereof (e.g., plasma or serum). Preferred samples are whole blood, serum, plasma, or urine. The sample can also be a partially purified fraction of a tissue or bodily fluid.

[0156] Reference sample can be a "normal" sample from a donor that does not have disease or condition body fluids, or from normal tissue in a subject that has disease or condition. Reference sample can also be from an untreated donor or cell culture that is not treated with active agent (e.g., no treatment or administration of vehicle only). Reference sample can also be taken at "time 0" before contacting cells or subject with the agent or therapeutic intervention to be tested, or at the beginning of a prospective study.

[0157] Exemplary types of non-cancerous (e.g., cell proliferative) diseases or disorders amenable to treatment with the selective degraders of EP300 of the present invention include inflammatory diseases and conditions, autoimmune diseases, neurodegenerative diseases, cardiac diseases, viral diseases, chronic and acute kidney disease or injury, metabolic diseases, and allergic and genetic diseases.

[0158] In some embodiments, the method is intended to treat subjects with cancer.Generally, the compounds of the present invention can be effective in treating carcinomas (solid tumors, including both primary and metastatic tumors), sarcomas, malignant melanomas, and hematological cancers (cancers that affect the blood, including lymphocytes, bone marrow and / or lymph nodes), such as leukemia, lymphoma, and multiple myeloma.Includes adult tumors / cancers and pediatric tumors / cancers.Cancers can be vascularized or not yet substantially vascularized or not vascularized tumors.

[0159] In some embodiments, the selective decomposition agent for EP300 of the present invention is effective for the treatment of cancers having dysregulation or dysfunction of EP300 (i.e., EP300-dependent cancers), such as NB, rhabdomyosarcoma, gastric cancer, brain cancer, pancreatic cancer, colorectal cancer (Gayther et al., Nat Genet 24:300-3 (2000)), breast cancer (Sobczak et al., Cancers (Basel) 11:1539 (2019)), lung cancer, lung squamous cell carcinoma, squamous cell carcinoma, prostate cancer, ovarian cancer, esophageal cancer, pancreatic cancer, retinoblastoma, cervical cancer, endometrial cancer, medulloblastoma, diffuse large B-cell lymphoma, acute lymphoblastic leukemia, bladder urothelial carcinoma, monocytic leukemia, squamous cell carcinoma of the head and neck (SCCHN), blood cancer, adult T-cell leukemia-lymphoma (ATLL), or NUT midline carcinoma.

[0160] Additionally, EP300 has been reported as a driver gene in bladder urothelial carcinoma where EP300 inhibition may be beneficial in addition to anti-PD-1 or anti-PD-L1 immunotherapy (Meng et al., Mol. Ther. Oncolytics 20:410-421 (2021); Chang et al., Exp. Mol. Med. 51:1-17 (2019)). In monocytic leukemia, the MLL-EP300 oncoprotein has been described, see Ohnishi et al., Eur. J. Haematol. 81:475-80 (2008). In SCCHN, a high CD8+ T-cell inflammatory phenotype is enriched in EP300 mutations (Saloura et al., Oral Oncol. 96:77-88 (2019)). In ATLL, 20% of cases with mutations in epigenetic and histone modifying genes had mutations in EP300 (Shah et al., Blood 132:1507-1518 (2018)). In NUT midline carcinoma, EP300 is involved in a feed-forward regulatory loop that leads to the propagation of oncogenic chromatin complex formation in bromodomain-containing protein 4 (BRD4)-NUT oncoprotein-induced cancer cells (Alekseyenko et al., Proc. Natl. Acad. Sci. USA 114:E4184-E4192 (2017)).

[0161] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual" 2nd Edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" and "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and therefore may be considered in making and practicing the invention. Particularly useful techniques for certain embodiments are described in the following sections.

[0162] The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and treatment methods of the present invention and are not intended to limit the scope of what the inventors regard as their invention. EXAMPLES

[0163] Example 1: Materials and Methods. The following materials and methods were utilized to obtain the results described herein. Chemical probes and biological reagents generated in this study are available for research purposes through a Material Transfer Agreement (MTA) or through commercial vendors. Data Availability and Experimental Model and Subject Details. RNA-seq and ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) database under SuperSeries accession number GSE159617, which consists of SuperSeries accession numbers GSE159613, GSE159614, GSE159615 and GSE159616. Code used in this study is described in the experimental details and is available upon request.

[0164] Cell lines. 293T, Kelly, BE2C, NGP, NB69 and SIMA neuroblastoma cell lines were obtained from the American Type Culture Collection (BE2C, 293T), the European Collection of Authenticated Cell Cultures (NB69), and the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (Kelly, NGP, SIMA). S2 cells were a gift from Dr. Karen Adelman (Harvard Medical School, Boston, MA). Cell lines used for exome-scale CRISPR-Cas9 screening and PRISM analysis are detailed in Corsello et al. Nat. Cancer 1:235-248 (2020) and Meyers et al. Nat. Genet. 49:1779-1784 (2017). All cell lines were short tandem repeat (STR) tested for identity. Neuroblastoma cell lines were cultured in Roswell Park Memorial Institute (RPMI) medium containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin and confirmed to be free of Mycoplasma species by routine testing.

[0165] Chemicals. Compounds C646 and CBP30 were obtained from Tocris™ Biosciences. Bortezomib, MLN4924, and thalidomide were obtained from Sigma-Aldrich®, and pomalidomide and lenalidomide were obtained from Target Molecule Corp. All other chemicals were synthesized and characterized in Qi Lab. Compounds JQAD1 and Biotin-JQAD1 were designed and synthesized based on the schemes listed in the following examples. The structures and purity of these compounds were further confirmed by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS). Animals. Eight-week-old female NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG™) mice (Jackson Laboratories, Catalog No.: 0005557) were used for tumor xenograft studies. For maximum tolerated dose testing, C57BL / 6-Crbn tm1.1Ble / J mice (Jackson Laboratories, Catalog No.: 032487) were used. For pharmacokinetic studies, Crl:CD1(ICR) mice (Charles River Laboratories, Catalog No. 022) were used. Further details including reagent or resource names, suppliers, and identifiers are listed in Table 2.

[0166] Quantitative and statistical analysis. Data from high-throughput sequencing coupled with chromatin immunoprecipitation (ChIP-Seq) and CRISPR-Cas9 screening were analyzed as described. Animal studies were analyzed by mixed-effects modeling and two-tailed analysis of variance (ANOVA) for tumor volume and weight means, and by log-rank test for survival. Other data were analyzed using one- or two-tailed ANOVA with post-hoc Tukey test, two-tailed t-test, or one-tailed Fisher exact test as appropriate for multiple or pairwise comparisons. Statistical significance was defined as p<0.05 unless otherwise stated. Data were analyzed with GraphPad Prism 7.01, and all error bars represent standard deviation unless otherwise stated.

[0167] Table 2. Key resources [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0168] Example 2: Cell viability assay. Cells were infected with lentivirus encoding sgRNA or treated with compounds as described. Colony assays were performed by reseeding cells at 500 cells per well in 6-well dishes and growing in regular growth medium for 10 days followed by 100% methanol fixation, 0.05% crystal violet staining, and quantification. Experiments were completed in triplicate; data shown are the average of three independent experiments. Cell Titer-Glo® assay was performed according to the manufacturer's instructions (Promega®). Briefly, 1000 cells / well were seeded in 96-well plates and treated with various compound doses. Cell viability was measured at the indicated time points based on luminescence by Cell Titer-Glo® assay (Promega®) and read on an Envision 2104 (PerkinElmer®, USA) according to the manufacturer's protocol.

[0169] Example 3: Propidium iodide-cell cycle analysis. Cells were infected with lentivirus encoding single guide RNA (sgRNA) or treated with compounds as described for the indicated length of time. Cells were then released from adhesion to the plate using a sterile spatula (Corning®), followed by centrifugation, aspirating the medium, and resuspending in hypotonic citrate-propidium iodide (PI) solution at 37°C for 30 minutes (Tate et al. Cytometry 4:211-215 (1983)). Nuclei were stabilized using 5M NaCl and then analyzed on a FACSAria™ II (BD Biosciences). Analysis of cell cycle phases was performed using FlowJo® v10.7 (BD Biosciences).

[0170] Example 4: Lentiviral infection. Stable and inducible cas9-expressing cell lines were generated using lentiviral particles produced in 293T cells. Briefly, lenticas9 (plasmid #52962), pCW-cas9-Blast (#83481), and pLKO.5-EGFP (#57822) plasmids were obtained from Addgene. Plasmids were transfected with pMD2.G (Addgene Plasmid #12259) and psPAX2 (#12260) using Lipofectamine 2000 (Invitrogen™) to generate viral particles by standard methodology. sgRNAs targeting individual genes were subcloned into pLKO.5-EFGP by standard methodology. Kelly, SIMA, BE2C, and NGP cells were infected with lenticas9 followed by blasticidin selection. Stable expression of cas9 was established by Western blotting of protein lysates with cas9 antibody (Cell Signaling Technology®). After infection with pLKO.5-EGFP-sgRNA lentivirus, cells were cultured for the specified times before evaluation. BE2C cells were infected with pIC-zsgreen or pIC-CRBN lentivirus and selected using 500 μg / mL hygromycin (Invitrogen™).

[0171] Example 5: Western blotting, immunoprecipitation and proteomic analysis. Cells growing in culture were lysed as described by Durbin et al. Nat. Genet. 50:1240-1246 (2018) and Wang et al. Nat. Commun. 10:5622 (2019). Nuclear lysates were prepared using the NE-PER® Nuclear Lysate Kit (Thermo Scientific™) according to the manufacturer's protocol. Chromatin lysates were prepared using a Total Histone Extraction Kit (Epigentek). Briefly, equal amounts of protein were separated by Western blotting using 4-12% Bis-Tris NuPAGE™ gels (Thermo-Fisher Scientific) prior to transfer and by immunoblotting using primary antibodies against: MYCN (1:1000, Cell Signaling Technology®), H3K27ac (1:1000, Abcam), total H3 (1:1000, Cell Signaling Technology®), EP300 (1:1000, Abcam), CBP (1:500, Cell Signaling Technology®), Cas9 (1:1000, Cell Signaling Technology®), cleaved PARP 1 (1:1000, Cell Signaling Technology), cleaved caspase-3 (1:1000, Cell Signaling Technology®), β-actin (1:1000, Cell Signaling Technology®), and β-actin (1:1000, Cell Signaling Technology®). Technology®), GATA3 (1:1000, EMD Millipore™), TFAP2β (1:1000, Cell Signaling Technology®), vinculin (1:1000, EMD Millipore™), CRBN (1:1000, Cell Signaling Technology®), HAND2 (1:1000, Santa Cruz Biotechnology).Secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse (1:5000, Santa Cruz Biotechnology) and incubated prior to exposure to enhanced chemiluminescence reagent (GE, Amersham). For immunoprecipitation, equal amounts of protein were diluted in buffer C as described in Mansour et al. Science 346:1373-1377 (2014) and incubated overnight with antibodies covalently coupled to Dynabeads™ M-270 beads (Thermo-Fisher Scientific) according to the manufacturer's instructions. Antibodies used included: H3K27ac, EP300 (Abcam), CBP, TFAP2β (Cell Signaling Technology®), rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology®). Immunoprecipitated proteins were isolated according to the manufacturer's instructions and subjected to Western blotting or mass spectrometry as described above.

[0172] Example 6: SILAC and H3K27ac co-IP mass spectrometry. To analyze the effect of JQAD1 on the nuclear proteome, Kelly cells were cultured in a 10-well plate. 13 C6 15 N2L-lysine and 13 C6 15 N4 cells were labeled with both L-arginine ("heavy" labeled cells) or normal L-lysine and L-arginine ("light" labeled cells). Heavy labeled cells were treated with 1 μM JQAD1 and light labeled cells were treated with an equal concentration of DMSO for 24 h before preparing nuclear lysates using the NE-PER® Nuclear Lysis Kit (Thermo Fisher Scientific). Untreated heavy and light cells were also lysed for nuclear proteins as controls. 750 μg of heavy and light nuclear lysates were pooled and subjected to trichloroacetic acid precipitation by standard methodology. Precipitated proteins were resuspended in 4× Laemmli sample buffer, boiled, and separated by SDS-PAGE by standard methodology. The gel was divided into two sections based on molecular weight and separated by 1 mm3The gel pieces were cut into pieces and subjected to a modified in-gel trypsin digestion procedure (Shevchenko et al. Anal. Chem. 68:850-858 (1996)). Briefly, the gel pieces were washed, dehydrated with acetonitrile, and rehydrated in 50 mM ammonium bicarbonate solution containing 12.5 ng / μl modified sequencing grade trypsin (Promega®) at 4° C. The samples were then washed and incubated in 50 mM ammonium bicarbonate solution at 37° C. for >16 h. Peptides were extracted by washing in 50% acetonitrile and 1% formic acid and dried by speed-vac. For analysis, samples were reconstituted in high performance liquid chromatography (HPLC) solvent A (2.5% acetonitrile, 0.1% formic acid) and loaded onto a nanoscale reversed-phase HPLC capillary column (2.6 μm C18 spherical silica beads in a fused silica capillary) detailed in Peng and Gygi, J. Mass. Spectrom. 36:1083-91 (2001). Samples were loaded via a FAMOS™ autosampler (LC Packings, San Francisco, CA). Peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid), subjected to electrospray ionization, and then entered into an LTQ Orbitrap Velos Pro™ ion trap mass spectrometer (Thermo Fisher Scientific). Peptides were detected, isolated, and fragmented to obtain tandem mass spectra of fragment ions specific to each peptide. Peptide sequences and protein identities were determined by matching the acquired fragmentation patterns against protein databases using Sequest® (Thermo Fisher Scientific) (Eng et al. J. Am. Soc. Mass Spectrom. 5:976-89 (1994)). All databases contained reversed versions of all peptide sequences, and data were filtered to a peptide false discovery rate of 1-2 percent. Processing was repeated three independent times and subjected to mass spectrometry three independent times.The total ratio of peptides and assigned proteins was used to calculate the change in abundance, comparing heavy peptides to light peptides in the 24 h (treated) samples and normalized to the 0 h control. Across three independent mass spectrometry assessments, 2493 proteins were detected and filtered for proteins present in detectable proportions at 0 h. Protein abundance was determined by Student's t-test, comparing 0 h abundance to 24 h abundance.

[0173] For co-immunoprecipitation / mass spectrometry of H3K27ac, BE2C and Kelly cells grown in normal growth medium were processed to collect nuclear lysates as described above. 750 μg of nuclear protein was immunoprecipitated as detailed above using Dynabeads™ M270 magnetic beads covalently coupled with H3K27ac antibody (Abcam) or normal rabbit IgG (Santa Cruz Biotechnology®) for more than 16 hours at 4°C, followed by washing and elution of immunoprecipitated proteins according to the manufacturer's instructions (Invitrogen™). Eluted proteins were subjected to trichloroacetic acid precipitation, trypsin digestion and mass spectrometry as described above. Two independent co-immunoprecipitation / mass spectrometry experiments were performed in each of BE2C and Kelly cells. In total, 366 and 281 proteins were identified to interact with H3K27ac and rabbit IgG in Kelly cells, and 1323 and 1113 proteins were identified in BE2C cells. Proteins identified by both H3K27ac and rabbit IgG were removed as non-specific binders, resulting in 167 and 492 protein interactors with H3K27ac in Kelly and BE2C cells, respectively. High confidence proteins were defined as the subset found in both Kelly and BE2C cells. This subset totaled n=35 proteins shown in Table 3, and gene identity and function were identified using Gene Ontology and PANTHER analysis (The Gene Ontology Consortium, Nucleic Acids Res 43:D1049-56 (2015); Mi et al. Nat. Protoc. 8:1551-1566 (2013)).

[0174] Table 3. H3K27ac-associated proteins identified by co-immunoprecipitation mass spectrometry [Table 3]

[0175] Table 3 shows proteins identified to interact with H3K27ac in both BE2C and Kelly cells, separated by co-immunoprecipitation / mass spectrometry. Normal rabbit IgG was used as a negative control. These high-confidence proteins were identified in two independent co-IP / mass spectrometry reactions per cell line and were found in both Kelly and BE2C cells, but not in the IgG control. Also shown is protein annotation by protein annotation through evolutionary relationship (PANTHER) protein class for each protein.

[0176] Example 7: In vivo studies. The protocol was approved by the Dana-Farber Cancer Institute Animal Care and Use Committee. Animals were maintained in accordance with the institution's guidelines. Eight-week-old female NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG™) mice (Jackson Laboratories, catalog #: 0005557) were used for tumor xenograft studies. For maximum tolerated dose studies, C57BL / 6-Crbn tm1.1Ble / J mice (Jackson Laboratories, catalog #: 032487) were used. For pharmacokinetic studies, Crl:CD1(ICR) mice (Charles River Laboratories, catalog # 022) were used.

[0177] For toxicity studies, four female CD1 mice (Charles River Laboratories) were intraperitoneally (IP) injected with a single dose of 10 mg / Kg (R,S)-JQAD1 solubilized in 10% hydroxypropyl β-cyclodextrin (Sigma-Aldrich®) in sterile water. After injection, blood concentrations of (R,S)-JQAD1 were measured by liquid chromatography with tandem mass spectrometry (LC-MS / MS) analysis with serial measurements at time points up to 24 hours. Pharmacokinetics was performed at ChemPartner, Shanghai, China using LC-MS / MS methodology, and pharmacokinetic parameters (T max , C max , T 1 / 2 , AUC, etc.) were calculated with WinNonlin® V 6.2 statistical software (Pharsight Corporation) using a non-compartmental model. For maximum tolerated dose (MTD) study, six female CD1 mice were treated with daily intraperitoneal administration of 10, 20, or 40 mg / kg (R,S)-JQAD1. Animals were monitored daily for body weight, grooming, and behavior, which had no significant effects. Humanized CRBN knock-in (Balb / c CRBN ILE391VAL For MTD study in the NIH (Jackson Laboratories), six mice per treatment group were treated with either vehicle control or 40 mg / kg / day (R,S)-JQAD1 by intraperitoneal injection. Animal weight, behavior, and grooming were monitored daily for a total of 21 days. On day 14, three mice per treatment group were sacrificed and tissues were fixed for immunohistochemistry. Blood was collected by retro-orbital puncture and blood tests were performed on a Hemavet® 9500FS (Drew Scientific) for blood count, creatinine, AST, ALT, ALP, GGTP, and BUN measurements at the Small Animal Imaging Facility at Beth Israel Deaconess Medical Center (Boston, MA).

[0178] For tumor studies, 8-week-old female NSG™ mice (Jackson Laboratories) were inoculated with 2.5×10 6 Kelly cells were implanted subcutaneously. Mice were assigned to three groups: vehicle (n=11), JQAD1 (40 mg / kg / day) (n=12), or JQAD1 (40 mg / kg, twice daily) (n=12) by intraperitoneal injection. Tumors engrafted and reached a size of 100–150 mm 3 When tumor size reached 100%, treatment with small molecule inhibitors was initiated. Mice were treated for 21 days and then followed for survival. Tumors were measured with calipers and mice were weighed every 3 days. Animals were euthanized according to institutional guidelines when tumor length or width reached 2,000 mm or when the animals became moribund. Tumor sizes were compared at each time point by two-way ANOVA with post-hoc Tukey test. Tumor growth curve kinetics were analyzed by both logistic regression and mixed-effects two-way ANOVA with post-hoc Tukey test to determine whether growth kinetics differed between treatment groups.

[0179] Separately, eight animals were xenografted as above and treated with vehicle (n=4) or JQAD1 (n=4) intraperitoneally at 40 mg / kg daily for 14 days. The animals were sacrificed on day 14, after which tumors were extracted and sectioned for immunohistochemical analysis or analysis of RNA expression by RNA sequencing (RNA-seq).

[0180] Example 8: Immunohistochemistry. Immunohistochemistry was performed on a Leica® Bond™ III automated staining platform. Antibody EP300, catalog number 86377, clone D8Z4E from Cell Signaling Technology® was run at 1:50 dilution using the Leica® Biosystems Refine Detection Kit with citrate antigen retrieval. Antibody KAT3A / CBP (catalog number ab2832, polyclonal, Abcam) was run at 1:200 dilution with ethylenediaminetetraacetic acid (EDTA) antigen retrieval using the Leica® Biosystems Refine Detection Kit.

[0181] Example 9: RNA-seq and analysis. For in vitro analysis, total RNA was extracted from control A485 or JQAD1-treated Kelly cells using TRIzol™ (Ambion). Prior to extraction, exogenous spike-ins of synthetic External RNA Control Consortium (ERCC) RNA controls were added based on cell number (Ambion). Samples were treated with RNase-free DNase I and spin-purified using the Qiagen® RNeasy Kit (Qiagen®). Purified RNA samples were subjected to library construction with polyadenylation preparation and sequencing using an Illumina NextSeq® 500 (paired-end, 75 bp reads).

[0182] RNA-seq reads were sorted using hisat 2.1.0 against a reference index containing the sequences of the hg19 amendment of the human reference genome and ERCC spike-in probes. Expression was quantified using the sorted BAM, gene references constructed using ERCC size, and RefSeq genes downloaded on July 15, 2017, and htseq-count with parameters "-I gene_id -stranded=reverse -f bam - m intersection-strict". Read counts were converted to transcripts per million (TPM) and used for cell number normalization. A pseudocount of 0.1 was added to all entries, with expression of all RefSeq genes and ERCC probes bounded at 0.01. Values ​​were normalized by balancing expression of ERCC probes between experiments using normalize.loess from the affy R package.

[0183] ERCC-normalized expression of each gene 24 hours after A485 or JQAD1 treatment was compared to its expression in dimethylsulfoxide (DMSO)-treated samples to generate a 2-fold change. These data were then analyzed by gene set enrichment analysis (GSEA) using the Gene Ontology Hallmark (H) collection in MSigDB to determine the relative enrichment for apoptosis hallmark gene sets in JQAD1 vs. A485-treated cells (The Gene Ontology Consortium, Nucleic Acids Res 43:D1049-56 (2015); Subramanian et al. Proc. Natl. Acad. Sci. USA 102:15545-50 (2005)).

[0184] For in vivo analysis, tumors were removed from animals treated with either vehicle phosphate buffered saline (PBS) and then filtered for single cells through a 0.45 micron filter. Single cell suspensions were then solubilized in TRIzol™ (Ambion) as described above and processed including ERCC RNA spike in controls, treatment with RNase-free DNase I, and spin purification. After preparation, there was enough material to proceed with RNAseq analysis for four vehicle control and three JQAD1-treated tumor specimens. Purified RNA samples were subjected to library construction with the low-input RNA protocol, followed by polyadenylation preparation and sequencing using an Illumina NextSeq® 500 (paired-end, 75 bp reads).

[0185] Raw reads for the in vivo model RNA-seq were first sorted using hisat2 v2.1 in paired-end mode against the mm9 modification of the mouse genome to filter contaminating mouse reads. The remaining reads were sorted against the reference genome, which contains the hg19 modification of the human reference and the sequence of the ERCC spike-in probe. Expression was quantified using the sorted BAM, gene references constructed using ERCC size, and RefSeq genes downloaded on 15 July 2017, and htseq-count with the parameters "-I gene_id -stranded=reverse -f bam -m intersection-strict". Read counts were converted to transcripts per million (TPM) and used for cell number normalization. Expression of all RefSeq genes and ERCC probes was bounded at 0.01, with a pseudocount of 0.1 added to all entries. Values ​​were normalized by balancing the expression of ERCC probes between experiments using normalize.loess from the affy R package.

[0186] Genes are then annotated as being regulated by either super-enhancers (n=671) or typical enhancers (n=27116) using H3K27ac data described in Durbin et al. Nat. Genet. 50:1240-6 (2018), Oldridge et al. Nature 528:418-21 (2015), and Wang et al. Nat. Commun. 10:5622 (2019) and available under GEO database accession number GSE94822. For annotation of gene identity as "transcription factor", a list of 1639 high-confidence human transcription factors was obtained from Lambert et al. Cell 175:598-9 (2018) and used to annotate the RNAseq data. Data were compared by ANOVA with multiple hypothesis testing using the original method of Benjamini and Hochberg to compare ERCC-regulated RNAseq expression in JQAD1-treated samples with vehicle-treated controls (Benjamini and Hochberg, Stat. Soc, Series B 57:289-300 (1995)).

[0187] Example 10: Biotin-JQAD1 pull-down assay. Biotin-JQAD1, synthesized below, was added to 500 μg of total Kelly cell lysate prepared in immunoprecipitation (IP) lysis buffer (Pierce™ Biotechnology) and incubated for 16 h at 4°C with end-over-end mixing. 100 μL of high capacity streptavidin agarose resin (Pierce™ Biotechnology) was loaded into a 1.5 mL Eppendorf tube® and washed three times with cold PBS before adding the cell lysate. The lysate was incubated at room temperature for 10 min, then centrifuged and washed, followed by elution in NuPAGE™ LDS sample buffer with reducing agent (Thermo-Fisher Scientific). Samples were processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described above and blotted using anti-EP300, anti-CRBN, or anti-CBP antibodies.

[0188] Example 11: AlphaLISA® Assay. The assay was performed with minimal modifications from the manufacturer's protocol (PerkinElmer®, USA). Briefly, 2x solutions of components with a final concentration of 50 nM CRBN-DDB1, 20 μg / ml Ni-coated acceptor beads, and 15 nM biotinylated pomalidomide were added in 10 μL to a 384-well plate (AlphaPlate-384, PerkinElmer®, USA). 100 nL of compounds in DMSO from stock plates were added by pin transfer using a Janus Workstation (PerkinElmer®, USA). Streptavidin-coated donor beads (final 20 μg / ml) were added to the solution followed by incubation at room temperature for 1 h and reading on an Envision 2104 (PerkinElmer®, USA) according to the manufacturer's protocol.

[0189] Example 12: Expression analysis of public databases. Analysis of publicly available expression datasets was performed using either the R2 database or the DepMap portal. A cancer cell line encyclopedia analysis of RNA expression and proteomic expression was performed using the 20Q1 data release (Ghandi et al. Nature 569:503-8 (2019); Nusinow et al. Cell 180:387-402 (2020)). The R2 database analysis was performed using the Kocak neuroblastoma dataset of n=649 primary tumor samples (Kocak et al. Cell Death Dis. 4:e586 (2013)).

[0190] Example 13: Motif enrichment analysis. CBP and EP300 ChIP-Seq peaks in Kelly and BE2C cells were compared to remove peaks bound by both factors. For each cell line, regions uniquely enriched for EP300 or CBP were determined using bedtools intersect -v -f 0.5 -r, which filters regions shared 50% or more between factors. These unique ChIP-seq peaks were: 7924 of 9274 peaks for EP300 and 717 of 2160 peaks for CBP in Kelly cells; 5679 of 8645 peaks for EP300 and 666 of 3732 peaks for CBP in BE2C cells. In each subset, motif enrichment analysis was performed as described in Mariani et al. Cell Syst. 5:187-201 (2017). Briefly, the 500 most-confident ChIP-seq narrow peaks, as assessed by FDR from peak calling, were identified and trimmed to 200 bp around the peak apex. A background set of 500 200 bp sequences, each corresponding to a trimmed peak, was generated using GENRE software with default human settings (promoter overlap, repeat overlap, GC content, CpG dinucleotide frequency). A collection of 44 position weight matrices (PWMs) was manually curated as a representative repertoire of sequence-specific transcription factor (TF) families, including motifs associated with previously determined master transcription factors in neuroblastoma cell lines (GATA3, TFAP2β, ISL1, MEIS2, PHOX2B, TCF3, and TWIST2). By comparing the trimmed peaks and associated background sequences, TF motif enrichment was quantified using the well-established area under the receiver operating curve (AUROC)-based metric that assesses the presence of the TF motif among the 500 highest confidence peaks (foreground set) compared to a background set of sequences (Gordan et al. Genome Res. 19:2090-2100 (2009)).For AUROC quantification, TF ChIP-seq data was analyzed as described by Mariani et al. Cell Syst. 5:187-201 (2017) (http: / / thebrain.bwh.harvard.edu / glossary-GENRE / download.html), including the use of the R function "matchPWM" (R package "Biostrings") to score each PWM against each sequence and the assessment of adjusted p-values ​​to ensure statistical significance. In both cell lines, TFAP2β (PWM M5912_1 from the CISBP databank version 1.02) was the only PWM that showed relevant differential enrichment, i.e., enrichment >0.6 AUROC in EP300-specific peaks (p-value <0.001) and no enrichment in CBP-specific peaks (AUROC ~0.5, p-value >0.1).

[0191] Example 14: Simultaneous relative inhibition profiling in mixtures (PRISM) screen. PRISM barcoded pool screening was performed using JQAD1 in 578 barcoded cell lines as described in Corsello et al. Nat. Med. 23:405-8 (2017) and Corsello et al. Nat Cancer 1:235-48 (2020). Some cell lines included in the screen were genetically engineered to express exogenous genes, and these cell lines were removed to obtain 557 cell lines. Briefly, cells in pools of 20–25 were thawed and seeded into 384-well plates (1250 cells / well for adherent cell pools and 2000 cells / well for suspension or mixed suspension / adherent cell pools) containing compounds (highest concentration: 10 μM, 8 points, 3-fold dilutions). All conditions were tested in triplicate. Cells were lysed 5 days after treatment and mRNA-based Luminex® detection of barcode abundance from lysates was performed as described in Corsello et al. Cancer 1:235-48 (2020). Luminex median fluorescence intensity (MFI) data was input into a standardized R pipeline to generate viability estimates relative to vehicle treatment for each cell line and treatment condition, and dose-response curves were fitted from the viability data.

[0192] Example 15: STRING database analysis. Neuroblastoma-specific genetic dependencies (n=146) were identified using the gene-ontology term "cellular component-nucleus" (Durbin et al. Nat. Genet. 50:1240-6 (2018)). Dependent genes were crossed with the Gene Ontology term "cellular component-nucleus" to obtain a list of dependent genes encoding nuclear factors (n=84). (The Gene Ontology Consortium, Nucleic Acids Res. 43:D1049-56 (2015); Mi et al. Nat. Protoc. 8:1551-66 (2013)). These genes were entered into the String database to generate interaction plots using medium-confidence interaction scores and hiding unconnected nodes. Network edges reflect evidence of interaction (Szklarczyk et al. Nucleic Acids Res. 43:D447-52 (2015)). Colors indicate commercially available compounds that target the protein (red=yes, grey=no).

[0193] Example 16: Genome-wide occupancy analysis. ChIP-seq was performed as previously described for cell lines (Durbin et al. Nat. Genet. 50:1240-6 (2018)). The following antibodies were used for ChIP: EP300 (Abcam, ab10485), CBP (#7389, Cell Signaling Technology®), TFAP2β (#2509, Cell Signaling Technology®), ASCL1 (sc-374104, Santa Cruz Biotechnology) and H3K27ac (Abcam ab4729). For each ChIP, 10 μg of antibody was added to 3 ml of sonicated nuclear extract. Illumina® sequencing, library construction, and ChIP-seq analysis methods were performed as described in Mansour et al. Science 346, 1373-7 (2014) and Sanda et al. Cancer Cell 22:209-21 (2012). The remaining ChIP-seq and assays for transposase-accessible chromatin (ATAC) sequencing data were extracted from previously published datasets available via the GEO portal (GSE120074, GSE94822, GSE65664). For experiments involving analysis of quantitative changes in H3K27ac, pellets of neuroblastoma cells were exogenously spiked with similarly fixed and treated S2 cells at a ratio of 1:10 prior to sonication.

[0194] Example 17: Cell line ChIP-seq and ATAC-seq processing and display. Reads were aligned to the human genome (hg19) using bowtie with parameters "-k 2 -m 2 -e 70-best and -l" set for read length. For visualization, WIG files were created from aligned ChIP-seq read positions using MACS 1.4 with parameters "-w -S -space=50 -nomodel -shiftsize=200" to artificially extend reads to 200 bp and calculate their density in 50 bp bins. Locus-specific visualization was performed using IGV 2.4.10 (Broad Institute).

[0195] Example 18: ChIP-seq enriched regions. Regions enriched for ChIP-seq signals were identified using MACS 1.4.2 with corresponding controls and parameters "- keep-dup=auto and -p 1e-9". Regions shown in Figure 2B, Figure 2C, Figure 9A, and Figure 9B were generated from collapsed conjugates of master transcription factor (HAND2, ISL1, PHOX2B, GATA3, TBX2, ASCL1, TFAP2β) peaks from each cell line.

[0196] Example 19: ChIP-seq coverage heat map. ChIP-seq and ATAC-Seq signals were quantified for heatmap display in 4 kb windows centered on the middle of each collapsed peak using bamToGFF with parameters "-m 50 -r -f 1". Rows were ordered by either MYCN signal (Figures 2C and 9B) or the ratio of EP300 to CBP in the entire displayed window. EP300 / CBP ratios were calculated in 500 bp windows centered on the middle of each collapsed master TF binding site using bamToGFF with parameters "-m 1 -r".

[0197] Example 20: ChIP-RX alignment and processing. ChIP-RX reads from Kelly cells treated with 500 nM JQAD1 were sorted in multiple steps. Reads were aligned to the dm6 revision of the Drosophila melanogaster reference genome using "-k 1 -chunkmbs 256 -- best" to identify spiked DNA. Fly read counts were determined by counting unique read names in the aligned read file. The remaining non-fly reads were aligned to the hg19 revision of the human reference genome using parameters "-k 2 -m 2 -chunkmbs 256 -best -l 75". Visualization files were constructed using macs 1.4 with parameters "-w -S -space=50 -nomodel -shiftsize=200" to generate wiggle files, which were then normalized by the millions of fly-mapped reads in the corresponding samples.

[0198] Example 21: Identification and assignment of super-enhancers and exemplary enhancers. Super-enhancers in Kelly xenografts were identified using ROSE and single-end BAMs generated as described above (Mansour et al. Science 346:1373-7 (2014)). Briefly, two sets of H3K27ac peaks were identified using MACS with the parameter sets "-keep-dup=auto -p 1e-9" and "-keep-dup=all -p 1e-9". The identified peak contacting the region chr2:14817188-17228298 was discarded as it falls within the MYCN surrounding genomic amplification region as described in Durbin et al. Nat. Genet. 50:1240-6 (2018)). The collapsed union of regions called using both MACS parameter sets that did not contact the discarded MYCN proximal region was used as input for ROSE, as described in Mansour et al. Science 346:1373-7 (2014), with some modifications. H3K27ac peaks were computationally stitched if they were within 12500bp of each other, whereas peaks that were entirely contained within + / - 2000bp of a RefSeq promoter were excluded from stitching. These stitched enhancers were ranked by their H3K27ac signal (length * density) minus the input signal. Super-enhancers were defined geometrically as enhancers above the point where the line y = x touches the curve. Stitched enhancers (typical and super-enhancers) were assigned to the single active gene whose transcription start site was closest to the center of the stitched enhancer. Active genes were determined by taking the top two-thirds of all RefSeq promoters (+ / -500bp) ranked by their H3K27ac signals. H3K27ac signals in promoters were determined using bamToGFF with parameters "-e 200 -m 1 -r -d".

[0199] We used bamToGFF with the parameter "-t TRUE" to quantify H3K27ac ChIP-RX read coverage of stitched enhancers, divided by millions of mapped reads, from which we subtracted the reads per million values ​​from the corresponding input experiments. These values ​​were used to create fold changes during the treatment time course.

[0200] Example 22: DepMap Dependency Analysis. Dependency data analysis was performed using the 20Q2 dataset retrieved from the DepMap portal. Dependency data was extracted as the probability of dependency for all cell lines (n=757) for two genes EP300 and CBP. Cell lines were annotated into lineages as described by the DepMap portal. Specific dependencies in neuroblastoma cell types were identified by extracting the probability of dependency on EP300 or CBP across 19 neuroblastoma cell types (SIMA, KPNYN, SKNDZ, SKNFI, CHP212, NB1, LS, Kelly, COGN305, COGN278, SKNBE2, LAN2, SKNAS, NGP, IMR32, GIMEN, NB1643, MHHNB11, CHLA15) and comparing with the probability of dependency >0.5 (Meyers et al. Nat. Genet. 49:1779-84 (2017); Oberlick et al. Cell Rep 28:2331-44 (2019)). For analysis across all tumor cell lines (Figure 7A-B), the mean and standard deviation of the probability of dependency were used as a continuous metric. Details of individual cell lines are available in the DepMap Portal.The lineages and numbers of cell lines in the 20Q2 DepMap release are as follows: myeloid leukemia (AML) (n=20), B-cell leukemia (n=11), lymphoma (n=19), bile duct / gallbladder cancer (n=29), bladder cancer (n=29), breast cancer (n=34), cervical cancer (n=12), sarcoma not otherwise specified (NOS) (n=8 - chondrosarcoma (n=1), epithelioid sarcoma (n=1), fibrosarcoma (n=2), leiomyosarcoma (n=1), pleomorphic sarcoma (n=1), thyroid sarcoma (n=1), undifferentiated sarcoma (n=1)), chordoma (n=3), chronic myeloid leukemia (CML) (n=7), colorectal cancer (n=37), endometrial / uterine cancer (n=26), Esophageal cancer (n=23), Ewing's sarcoma (n=15), eye cancer (n=4), gastric cancer (n=26), glioblastoma (n=33), glioma (n=17), head and neck cancer (n=32), renal cancer (n=21), liposarcoma (n=5), liver cancer (n=22), lung cancer (n=106), medulloblastoma (n=8), malignant melanoma (n=41), multiple myeloma (n=20), neuroblastoma (n=19), osteosarcoma (n=8), ovarian cancer (n=42), pancreatic cancer (n=34), rhabdoid tumor (n=7), rhabdomyosarcoma (n=11), squamous cell carcinoma (n=4), synovial sarcoma (n=5), T-acute leukemia (n=3) and thyroid cancer (n=6). Data from lines with fewer than three cell lines were removed from the analysis.

[0201] Example 23: Synthesis of JQAD1 [ka]

[0202] Scheme 1: Synthesis of JQAD1 (12-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)-N-((R)-3'-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidine]-5-yl)dodecanamide). Compounds Int-1 and Int-2 are described in Michaelides et. al., ACS Med. Chem. Lett. 9:28-33 (2018) and International Patent Publication WO2020 / 006157 A1. To a mixture of Int-1 (500 mg, 1.04 mmol, 1.0 equiv.) and Int-2 (492 mg, 1.04 mmol, 1 equiv.) in N,N-dimethylformamide (DMF, 10 mL, 0.1 M) in a 50 mL flask, N,N-diisopropylethylamine (DIPEA) (349 μL, 2.09 mmol, 2 equiv.) and hexafluorophosphate azabenzotriazole tetramethyluronium (HATU) (793 mg, 2.09 mmol, 2 equiv.) were added. The reaction mixture was stirred at 25 °C for 2 h. After the reaction was complete, the mixture was directly purified by silica gel chromatography (ethyl acetate / hexane, 20-90% gradient) and the solvent was removed under reduced pressure to give JQAD1 as a yellow powder (700 mg, 72% yield).

[0203] 1HNMR (500 MHz, アセトン-d6) i 9.90 (d, J = 4.0 Hz, 1H), 9.26 (d, J = 5.1 Hz, 1H), 7.90 (d, J = 7.7 Hz, 1H), 7.59 (td, J = 7.8, 2.7 Hz, 1H), 7.49 (d, J = 9.7 Hz, 3H), 7.36 - 7.31 (m, 1H), 7.22 (t, J = 8.6 Hz, 2H), 7.13 - 7.00 (m, 3H), 6.42 (d, J = 5.9 Hz, 1H), 5.51 (p, J = 7.8 Hz, 1H), 5.07 (ddd, J = 12.1, 7.6, 4.2 Hz, 2H), 4.97 - 4.81 (m, 1H), 4.67 (dd, J = 71.1, 16.7 Hz, 1H), 4.43 (dd, J = 90.6, 16.6 Hz, 1H), 3.38 (q, J = 6.3 Hz, 2H), 3.28 - 3.04 (m, 2H), 3.03 - 2.85 (m, 3H), 2.85 - 2.70 (m, 4H), 2.56 (dddd, J = 14.5, 12.1, 8.6, 4.2 Hz, 1H), 2.39 (t, J = 7.2 Hz, 2H), 2.27 - 2.17 (m, 1H), 2.07 (p, J = 2.2 Hz, 2H), 1.73 - 1.67 (m, 4H), 1.39 (dd, J = 38.4, 5.3 Hz, 12H).

[0204] MS (ESI) calculated value. C 48 H 52 F4N6O9 について: 932.37, measured value: [M+1] 933.36.

[0205] Example 24: Synthesis of ビオチン-JQAD1.

change

[0206] Int-3 ((9H-fluoren-9-yl)methyl tert-butyl(6-((((R)-3'-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidine]-5-yl)methyl)amino)-6-oxohexane-1,5-diyl)dicarbamate).

[0207] To a solution of Int-1 (20.0 mg, 0.042 mmol, 1 equiv.), Boc-Lys(Fmoc)-OH (19.7 mg, 0.042 mmol, 1 equiv.) in DMF (3 mL, 0.14 M), DIPEA (14.0 μL, 0.084 mmol, 2 equiv.) and HATU (31.9 mg, 0.084 mmol, 2 equiv.) were added. The reaction mixture was stirred at 25 °C for 2 h. After the reaction was complete, the mixture was directly purified by silica gel chromatography (ethyl acetate / hexane, 20-90% gradient) and the solvent was removed under reduced pressure to give int-3 as a yellow powder (33.7 mg, 85% yield).

[0208] MS(ESI) calculated value. C 50 H 53 For F4N5O9: 943.38, measured value: [M+1] 944.39. [ka]

[0209] Scheme 2: Synthesis of biotin-JQAD1 (6-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)hexanamide)-N-((R)-3'-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidine]-5-yl)-2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide)hexanamide). [ka]

[0210] Int-4 (tert-butyl (6-amino-1-((((R)-3'-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidine]-5-yl)methyl)amino)-1-oxohexan-2-yl)carbamate)

[0211] To a solution of Int-3 (33.7 mg, 0.036 mmol) in dichloromethane (DCM) (2 mL, 0.018 M) was added diethylamine (1 mL) dropwise. The reaction was stirred at 25 °C for 1 h. The solvent was removed under reduced pressure and the resulting residue was purified by silica gel chromatography (MeOH / DCM, 0-10% gradient). The solvent was removed under reduced pressure to give Int-4 as a colorless oil (25.4 mg, 95% yield).

[0212] MS(ESI) calculated value. C 35 H 43 For F4N5O7: 721.31, measured value: [M+1] 722.35.

[0213] Int-5 was synthesized according to WO 2020 / 006157 A1. [ka]

[0214] Int-6 (tert-butyl (6-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)hexanamide)-1-(((R)-3'-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidine]-5-yl)amino)-1-oxohexan-2-yl)carbamate)

[0215] To a solution of Int-4 (20.0 mg, 0.028 mmol, 1 equiv.) and Int-5 (11.9 mg, 0.028 mmol, 1 equiv.) in DMF (2 mL, 0.014 M), DIPEA (9.33 μL, 0.056 mmol, 2 equiv.), HATU (21.3 mg, 0.084 mmol, 2 equiv.) were added. The reaction mixture was stirred at 25 °C for 2 h. After the reaction was complete, the mixture was purified by silica gel chromatography (MeOH / DCM, 0-10% gradient) and the solvent was removed under reduced pressure to give Int-6 as a yellow oil (21.1 mg, 70% yield). [ka]

[0216] Int-7 (2-amino-6-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)hexanamide)-N-((R)-3'-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidine]-5-yl)hexanamide).

[0217] To a solution of Int-6 (21.1 mg, 0.020 mmol) in DCM (2 mL, 0.01 M) was added trifluoroacetic acid (TFA) (1 mL) dropwise. The reaction was stirred at 25° C. for 1 h and the solvent was removed under reduced pressure. The resulting residue was subjected to the next step reaction without further purification. [ka]

[0218] To a solution of Int-7 and biotin (2.45 mg, 0.010 mmol, 1 equiv.) in DMF (1 mL, 0.01 M), DIPEA (3.33 μL, 0.020 mmol, 2 equiv.) and HATU (7.51 mg, 0.020 mmol, 2 equiv.) were added. The resulting mixture was stirred at 25 °C for 2 h. After the reaction was complete, the mixture was purified by silica gel chromatography (MeOH / DCM, 0-10% gradient) and the solvent was removed under reduced pressure to give biotin-JQAD1 as a yellow oil (5.3 mg, 52% yield).

[0219] MS(ESI) calculated value. C 58 H 66 F4N 10 O 12 For S: 1202.45, actual value: [M+1] 1023.48.

[0220] Other embodiments Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0221] The patents and scientific literature referred to herein establish knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. Genbank and NCBI deposits indicated by accession numbers cited herein are incorporated by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated by reference.

[0222] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. 1. A selective degrader of E1A binding protein P300 (EP300) for use in a method of treating a subject having a disease or disorder associated with EP300 dependence, said method comprising: obtaining a test sample from a subject having or at risk of developing said disease or disorder; identifying an increased expression level of cereblon (CRBN) in the test sample compared to the expression level of CRBN in a reference sample; and The method includes administering a selective decomposition agent for EP300 to the subject; Selective decomposition of EP300 for use.

2. The test sample is obtained from a tumor tissue or tumor microenvironment; or The test sample is obtained from a bodily fluid selected from the group consisting of plasma, blood, urine, sputum, and cerebrospinal fluid (CSF); or The selective decomposition agent for EP300 for use according to claim 1, wherein the reference sample is obtained from healthy normal tissue or tumor tissue.

3. A selective decomposition agent of EP300 for use as described in claim 2, wherein the reference sample is obtained from healthy normal tissue from the same individual as the test sample or one or more healthy normal tissues from a different individual.

4. The selective decomposition agent for EP300 for use according to claim 1, wherein the selective decomposition agent for EP300 is JQAD1 or a pharma- ceutically acceptable salt thereof. 【Chemistry 1】

5. The selective decomposition agent for EP300 for use according to claim 1, wherein the disease or disorder is EP300-dependent cancer.

6. the cancer involves a solid tumor, or The selective decomposition agent for EP300 for use according to claim 5, wherein the cancer is a blood cancer.

7. the solid tumor is neuroblastoma, rhabdomyosarcoma, malignant melanoma, colon cancer, rectal cancer, gastric cancer, breast cancer, brain cancer, or pancreatic cancer; or The selective decomposition agent for EP300 for use according to claim 6, wherein the blood cancer is leukemia, myeloma, or lymphoma.

8. The selective decomposition agent for EP300 for use according to claim 7, wherein the neuroblastoma is high-risk neuroblastoma.

9. tumor cell survival, tumor cell proliferation, or tumor metastasis is inhibited, or A selective decomposition agent for EP300 for use according to claim 5, wherein tumor cell proliferation is reduced or tumor cell apoptosis is induced.

10. further comprising administering to the subject a chemotherapy agent, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or stem cell transplant; or or further comprising administering to the subject a combination chemotherapeutic agent; The selective decomposition agent for EP300 for use according to claim 1, further comprising administering an immunosuppressant to the subject.

11. the chemotherapeutic agents include cis-retinoic acid, cyclophosphamide, cisplatin, carboplatin, vincristine, doxorubicin, etoposide, topotecan, busulfan and melphalan, or thiotepa; or The selective decomposition agent for EP300 for use according to claim 10, wherein the chemotherapeutic agent is administered together with a steroid.

12. The selective decomposition agent for EP300 for use according to claim 11, wherein the steroid comprises prednisone or dexamethasone.

13. The selective degrading agent of EP300 for use according to claim 10, wherein the combination chemotherapy agent comprises carboplatin or cisplatin, cyclophosphamide, doxorubicin, and etoposide, or irinotecan, temozolomide, or ifosfamide.

14. The selective decomposition agent for EP300 for use according to claim 10, wherein the immunosuppressant comprises dinutuximab with or without cis-retinoic acid.

15. The selective decomposition agent for EP300 for use according to claim 1, wherein the subject is a human.

16. The selective decomposition agent for EP300 for use according to claim 1, wherein a therapeutically effective amount of the selective decomposition agent for EP300 or a pharma- ceutically acceptable salt thereof is orally administered to a subject in the form of a tablet.

17. The selective decomposition agent of EP300 for use according to claim 1, wherein a therapeutically effective amount of the selective decomposition agent of EP300 or a pharma- ceutically acceptable salt thereof is orally administered to a subject in the form of a capsule.

18. The selective decomposition agent of EP300 for use according to claim 1, wherein a therapeutically effective amount of the selective decomposition agent of EP300 or a pharma- ceutically acceptable salt thereof is parenterally administered to a subject in liquid form.

19. A method for determining whether EP300 degradation in a subject with cancer provides a clinical benefit in said subject. Determining the expression level of CRBN in a test sample obtained from a subject having or at risk of developing cancer; comparing the expression level of CRBN in the test sample to the expression level of CRBN in a reference sample; and determining whether EP300 degradation inhibits the cancer in the subject if the expression level of CRBN in the test sample differs from the expression level of CRBN in the reference sample.

20. The test sample is obtained from a tumor tissue or tumor microenvironment; or 20. The method of claim 19, wherein the test sample is obtained from a bodily fluid.

21. the bodily fluid is selected from the group consisting of plasma, blood, urine, sputum, and CSF; or 20. The method of claim 19, wherein the reference sample is obtained from healthy normal tissue.

22. the clinical benefit in the subject comprises a complete or partial response as defined by Response Evaluation Criteria in Solid Tumors (RECIST), stable disease as defined by RECIST, or long-term survival despite disease progression or response as defined by irRC criteria; or or determining that EP300 degradation in a subject with cancer provides a clinical benefit in the subject when the test sample is obtained from the cancer and the expression level of CRBN in the test sample is equal to or greater than the level of CRBN in the reference sample; or 20. The method of claim 19, further comprising determining that EP300 degradation in a subject having cancer does not provide clinical benefit in the subject when the test sample is obtained from the cancer and the expression level of CRBN in the test sample is lower than the level of CRBN in the reference sample.

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