Cancer diagnosis and treatment using PRMT5 inhibitors

Abstract

The present invention relates to a method for patient selection and for treating cancer with a protein arginine methyltransferase 5 (PRMT5) inhibitor, wherein the expression levels of retinoblastoma tumor suppressor protein (pRb) and / or total E2F1 protein in a cancer cell-containing biological sample from a patient are used to determine whether the patient should be treated with a PRMT5 inhibitor.

Description

[Technical field]

[0001] The present invention relates to the field of cancer. In particular, it relates to a patient selection method and a method for treating cancer with a PRMT5 inhibitor. The present invention also provides a kit for use in the method of the present invention. [Background technology]

[0002] The retinoblastoma protein (pRb)-E2F pathway is a keystone of cell cycle control. It is often deregulated in tumor cells, and deregulation of this pathway is widely considered a "hallmark" of cancer. Classically, the pRb tumor suppressor protein is a negative regulator of E2F transcription factors that act as transcriptional pivots through which pRb exerts its effects on cell cycle progression.

[0003] PRMT5 has been identified as a therapeutic target for the treatment of cancer. Indeed, many patent publications disclose PRMT5 inhibitors and their use in treating cancer.

[0004] However, cancer is a heterogeneous disease, and among patients with a certain type of cancer (e.g., breast cancer or lung cancer), there are cancer subpopulations. It is widely recognized that no drug is effective in treating everyone with a certain type of cancer, but the advent of personalized medicine, which can identify patients who are likely to respond well to a certain type of drug, for example by using diagnostic tests for gene mutations characteristic of response, means that those who are likely to respond to a drug can be identified and treated, and those who are unlikely to respond can be excluded from treatment with drugs that are unlikely to be effective but probably cause toxic side effects. These unlikely responders can therefore be protected from inappropriate treatment and can be treated in some other manner.

[0005] There is an ongoing need, particularly in the oncology field, for means to identify patients who will respond, or are likely to respond, to a particular type of treatment.

[0006] E2Fs are a family of transcription factors that have been implicated in various cell fates, including proliferation, apoptosis, and differentiation (Stevens and La Thangue, 2003; Frolov and Dyson 2004; Polager and Ginsberg 2008; van den Heuvel and Dyson 2008). E2F proteins share the ability to regulate a diverse set of target genes (Frolov and Dyson 2004; van den Heuvel and Dyson 2008). The first family member identified, E2F-1, physically interacts with the retinoblastoma tumor suppressor protein pRb, which negatively regulates E2F-1 activity (Bandara and La Thangue 1991; Zamanian and La Thangue 1992; Weinberg 1995; Stevens and La Thangue 2003). While it is established that E2F-1 can promote proliferation, it is also becoming clear that E2F-1 can induce apoptosis (van den Heuvel and Dyson 2008; Polager and Ginsberg 2008). In Rb- / - mice, high levels of apoptosis in certain tissues reflect deregulated E2F-1 activity (Tsai et al. 1998; Iaquinta and Lees 2007). Furthermore, E2F-1- / - mice suffer from high incidence of tumors (Field et al. 1996), suggesting that E2F-1 assumes a tumor suppressor role in some tissues, possibly reflecting its ability to induce apoptosis. However, the mechanisms by which E2F-1 activity, particularly its apoptotic activity, influences the diverse cellular outcomes that are believed to be due to the cell environment dependency of these events remain elusive. It is the purpose of the present invention to identify such mechanisms.

[0007] E2F-1 is regulated not only during cell cycle progression (Stevens and La Thangue, 2003; van den Heuvel and Dyson 2008) but also under conditions of DNA damage (Pediconi et al. 2003; Stevens et al. 2003; Stevens and La Thangue 2003). In cells with DNA damage, E2F-1 is induced in a manner that follows kinetics similar to p53 (Pediconi et al. 2003; Stevens and La Thangue 2003), which coincides with the activation of a diverse collection of E2F target genes (Ren et al. 2002). DNA damage activates signaling pathways involving protein phosphokinases such as ATM / ATR and Chkl / Chk2, which in turn phosphorylate effector proteins that mediate the outcome of the DNA damage response (Jackson and Bartek, 2009). Both families of DNA damage response kinases phosphorylate E2F-1, which contributes to its regulation in cells with DNA damage (Stevens et al. 2003; Stevens and La Thangue 2003). Furthermore, E2F-1 induces apoptosis under DNA damaging conditions and may provide an important pathway allowing apoptosis to be activated in tumor cells where p53 activity is compromised (Stevens and La Thangue 2003).

[0008] The retinoblastoma tumor suppressor protein (pRb) is a key regulator of the G1 to S phase transition during the cell cycle, a process essential for precisely controlled cell proliferation (Dick and Rubin, 2013). pRb / Rb1 loss is common in all cancer types, and it is strongly associated with poor overall survival (Knudsen et al., 2020; and Ertel et al., 2010). Loss of Rb in cancer can occur through deletion of one or both copies of the gene, mutations resulting in nonfunctional proteins, or promoter methylation of the Rb gene (Hanahan and Weinberg, 2000). In addition to mutations in the RB1 gene itself, upstream Rb pathway aberrations can occur in a high percentage of human cancers, for example, inactivation of CDK inhibitors and activation of cyclins and CDKs are frequent events (Hanahan and Weinberg, 2000).

[0009] PRMT5 is a member of the protein arginine methyltransferase (PRMT) family. PRMT5 is a type II methyltransferase that catalyzes the symmetric demethylation of its substrate proteins (Bedford and Clarke et al., 2009). Arginine methylation is known to play a role in a wide variety of cellular processes. In recent years, an increasing number of reports have highlighted the potential role of PRMT5 as a carcinogen (Jarrold and Davis, 2019; Kim and Ronai, 2020). PRMT5 has been shown to promote tumor development in a wide range of cancers, including hematological, colon, breast, prostate, lung, liver, bone, skin, ovarian, gastric, brain, and pancreatic cancers (Shailesh et al., 2018).

[0010] E2F-1 has been shown to be a target for methylation by PRMT5, a protein arginine methyltransferase. Methylation of E2F1 by PRMT5 promotes cell proliferation by increasing the transcription of cell cycle-related E2F1 target genes (Cho et al., 2012; and Zheng et al., 2013). Furthermore, a subset of genes under the control of E2F1 and PRMT5 have been identified that may affect cancer cell viability, migration, invasion, and adhesion (Barczak et al., 2020). Co-occurrence of high levels of expression of both E2F1 and PRMT5 in several cancers is common, and this has been found to correlate with poor prognosis (Barczak et al., 2020).

[0011] There is a need in the art for methods that can select patients most likely to respond well to cancer treatment with PRMT5 inhibitors. The present invention addresses that need. Summary of the Invention

[0012] According to a first aspect of the invention there is provided a method of selecting a treatment for a patient with cancer comprising the steps of: (i) determining the expression level of biomarkers (a) pRb and / or (b) total E2F1 in a cancer cell-containing biological sample from a patient; (ii) comparing the expression levels in (i) with a reference value for each biomarker; (iii) if the patient's cancer cells exhibit decreased expression of pRb and / or increased expression of total E2F1 compared to baseline, the patient is selected for treatment with a PRMT5 inhibitor; A method is provided.

[0013] The expression level of pRb can be determined by quantifying the amount of pRb protein or the amount of RB1 transcript in a sample. The expression level of total E2F1 can be determined by quantifying the amount of total E2F1 protein or the amount of E2F1 transcript in a sample.

[0014] Total E2F-1 refers to the sum of all types of E2F-1, not just one form of E2F-1, such as methylated E2-F1.

[0015] According to a second aspect of the present invention, there is provided a method of selecting a treatment for a patient having cancer, comprising the step of determining whether the PRB1 gene in the cancer cells contains one or more mutations that result in reduced expression (including null) of pRb, and if the cancer cells contain one or more mutations in the RB1 gene that result in reduced expression of pRb, then the patient is selected for treatment with a PRMT5 inhibitor.

[0016] According to a third aspect of the invention there is provided a kit for use in the method of the first aspect of the invention comprising one or more reagents capable of determining the expression level of pRb and / or total E2F1.Suitably the kit comprises an antibody or antigen-binding portion thereof which specifically binds to E2F-1 protein, and / or an antibody or antigen-binding portion thereof which specifically binds to pRb protein, and / or a nucleic acid oligonucleotide capable of specifically binding to the RB1 transcript, and / or a nucleic acid oligonucleotide capable of specifically binding to the E2F1 transcript.

[0017] According to a fourth aspect of the invention there is provided a PRMT5 inhibitor for use in treating a patient identified according to the first aspect of the invention.

[0018] In particular, according to a variant of the fourth aspect of the present invention, there is provided a PRMT5 inhibitor for use in treating cancer in which cells express higher than normal levels of total E2F1 protein and / or reduced levels of pRb protein compared to normal.

[0019] According to a fifth aspect of the invention there is provided a PRMT5 inhibitor for use in the treatment of a pRb deficient cancer or for use in the treatment of a cancer whose cells express reduced levels of the pRb biomarker compared to normal.

[0020] Suitably, a pRb deficient cancer is a cancer comprising cancer cells which contain one or more mutations in the RB1 gene that result in reduced expression (including no expression) of pRb in the cancer cells.

[0021] Any agent capable of inhibiting PRMT5 may be utilized in the fourth or fifth aspect of the present invention. Suitably, the PRMT5 inhibitor is a small molecule compound, an antisense oligonucleotide, an RNAi molecule, or an antibody or binding fragment thereof.

[0022] According to a sixth aspect of the present invention there is provided a computer implemented method for assisting in selecting a treatment for a patient with cancer, comprising the steps of: (i) receiving a value for the level of biomarkers (a) pRb and / or (b) total E2F1 in a cancer cell-containing biological sample from a patient; (ii) comparing the levels in (i) to a reference value for each biomarker; (iii) if the patient's cancer cells exhibit a decrease in the level of pRb and / or an increase in the level of total E2F1 compared to baseline, the patient is selected for treatment with a PRMT5 inhibitor; A method is provided.

[0023] Preferably, the level is an expression level.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, but preferred methods and materials are described below.In case of conflict, the present specification contains definitions and takes precedence.In addition, materials, methods and examples are merely illustrative and are not intended to be limiting.

[0025] Other features and advantages of the present invention will become apparent from the following description. [Brief description of the drawings]

[0026] [Figure 1] Figure 2: PRMT5 inhibition mediates cancer cell death and this effect is E2F1 dependent. U2OS cells were transfected with PRMT5 (P5), E2F-1 (E2F1) or control (NC) or siRNA as indicated, and colony growth was measured on day 10 after staining with crystal violet. (a) The levels of E2F1 and PRMT5 in siRNA-treated cells are shown by Western blot (b). [Diagram 2] Figure 1 shows that cells with a genetic deletion of E2F1 are less sensitive to PRMT5 inhibition: (a) Cell viability assay in T-47D cell line (breast cancer), (b) Cell viability assay in HCT116 cell line (colorectal cancer). [Diagram 3] Figure 1 shows that cells with genetic deletion of pRb are more sensitive to PRMT5 inhibition: (a) Cell viability assay in T-47D cell line (breast cancer), (b) Cell viability assay in U2OS cell line (bone cancer). [Figure 4] Figure 1 shows that cells with a genetic deletion of E2F1 are less sensitive to the PRMT5 inhibitor GSK3326595: (a) Cell viability assay in HCT116 cell line (colorectal cancer), (b) Cell viability assay in T-47D cell line (breast cancer). [Diagram 5] FIG. 1 shows that cells with a genetic deletion of E2F1 are less sensitive to the PRMT5 inhibitor PF06939999. (a) Cell viability assay in HCT116 cell line (colorectal cancer), (b) Cell viability assay in T-47D cell line (breast cancer). [Figure 6] Figure 1 shows cell viability assay in HCT116 cell line (colorectal cancer). Cells with genetic deletion of E2F1 are less sensitive to the PRMT5 inhibitor JNJ64619178. [Figure 7] Figure 1 shows cell viability assay in HCT116 cell line (colorectal cancer). Cells with a genetic deletion of E2F1 are less sensitive to the PRMT5 inhibitor LLY283. [Figure 8] Figure 1 shows cell viability assay in T-47D cell line (breast cancer). Cells with genetic deletion of pRb are more sensitive to the PRMT5 inhibitor GSK3326595. [Figure 9] Figure 1 shows cell viability assay in T-47D cell line (breast cancer). Cells with genetic deletion of pRb are more sensitive to the PRMT5 inhibitor PF06939999. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The disclosed method may be more readily understood by reference to the following detailed description, which forms a part of this disclosure. It should be understood that the disclosed method is not limited to the specific method described and / or shown herein, and that the terminology used herein is merely for the purpose of describing specific embodiments by way of example, and is not intended to be a limitation of the claimed method.

[0028] The methods of the present disclosure employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry that are within the skill of the art. Exemplary techniques are described in "Molecular Cloning: A Laboratory Manual", 2 nd edition (Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989); “Current Protocols in Molecular Biology” (FM Ausubel et al., eds., Current Protocols of Molecular Biology, John Wiley and Sons (1987); and “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds., Birhauser, Boston, 1994).

[0029] In describing the present invention, the following terms will be employed and are intended to be defined as set forth below.

[0030] It should be understood that certain features of the disclosed methods, which are, for clarity, described herein in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any subcombination.

[0031] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.

[0032] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0033] The term "and / or" should be understood to mean either one or both of the alternatives.

[0034] As used herein, and unless otherwise indicated, the term "about" should be understood to be used synonymously with the term "approximately". Illustratively, and unless otherwise indicated, the use of the term "about", when used with a given numerical value or range, represents something more or something less than the given value or range, up to within ±15% of the given value or range, ±10% of the given value or range, ±5% of the given value or range, or conveniently ±2% of the given value or range. Thus, such values ​​are encompassed by the claims reciting the term "about" or "approximately".

[0035] As used herein, "cancer sample" or "cancer cell-containing sample" refers to any biological sample that contains one or more cancer cells, or one or more cancer-derived RNA or protein, and is obtained from a cancer patient.For example, the tissue sample obtained from the cancer tissue of a cancer patient is a cancer sample that is useful in the present invention.The tissue sample may be a formalin-fixed paraffin-embedded (FFPE) sample, or a fresh frozen sample, and preferably contains a majority of cancer cells.

[0036] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an agent that confers a therapeutic effect on the treated subject at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject shows or feels an effect). In particular, an "effective amount" refers to an amount of a therapeutic agent that is effective to treat, ameliorate or prevent a desired disease or condition, or to exhibit a detectable therapeutic or prophylactic effect, such as by improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease.

[0037] The effective amount is usually administered in a dosing regimen that may include multiple unit doses. For any particular therapeutic agent, the effective amount (and / or the appropriate unit dose within an effective dosing regimen) may vary, for example, depending on the route of administration, combination with other drugs. Also, the specific effective amount (and / or unit dose) for any particular patient may depend on a variety of factors, including which disorder is being treated; the severity of the disorder; the activity of the specific drug used; the specific composition used; the age, weight, general health and diet of the patient; the time of administration; the route of administration; the duration of treatment, etc., as is well known in the medical field. The therapeutically effective amount is typically the dosage of the drug approved by public health authorities (e.g., the U.S. Food and Drug Administration [FDA] or the European Medicines Agency [EMA]) as determined from controlled human clinical trials.

[0038] The term "inhibitor" as used herein refers to an entity / agent whose presence in a system in which an activity of interest is observed correlates with a decrease in the level and / or nature of that activity compared to the level and / or nature of that activity observed in the absence of the inhibitor under otherwise comparable conditions. In some embodiments, the inhibitor interacts directly with the target whose activity is of interest. In some embodiments, the inhibitor affects the amount / level of the target of interest, or alternatively or in addition, in some embodiments, the inhibitor affects the activity of the target of interest without affecting the level of the target. In some embodiments, the inhibitor affects both the level and activity of the target entity of interest, such that the observed difference in activity is not fully explained or commensurate with the observed difference in level. The inhibitor may be any agent, e.g., a small molecule compound, a nucleic acid, an antibody, etc. The target may be a protein or precursor thereof, or a nucleic acid, e.g., genomic DNA or mRNA, encoding said protein / precursor.

[0039] The term "oligonucleotide" refers to relatively short polynucleotides, including, but not limited to, single-stranded deoxyribonucleotides, single-stranded or double-stranded ribonucleotides, RNA / DNA hybrids, and double-stranded DNA. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example, using commercially available automated oligonucleotide synthesizers. However, oligonucleotides can be produced by a variety of other methods, including in vitro recombinant DNA-mediated techniques, and by expressing DNA in cells and organisms.

[0040] The term "polynucleotide", when used in singular or plural, generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.

[0041] As used herein, the term "primer" refers to a molecule, typically a single-stranded oligonucleotide, that can be used to generate a PCR reaction amplification product (amplicon). A primer may also be referred to as a PCR primer.

[0042] As used herein, the term "probe" refers to a molecule, typically a single-stranded oligonucleotide, that can be used to detect a complementary target nucleic acid product by hybridizing to it due to sequence complementarity. Probes may be labeled, such as with a fluorescent marker or other label, to facilitate detection. Probes may also be referred to as hybridization primers.

[0043] The term "RNA transcript" as used herein refers to the RNA transcription products of a gene, including, for example, mRNA, unspliced ​​RNA, splice variant mRNA, microRNA, and fragmented RNA.

[0044] As used herein, "selectively hybridize" means that it can only hybridize to a single complementary region that is unique to the target in a sample, for example, under conditions that allow selective hybridization to target nucleic acid.In this way, each primer or probe can only hybridize to one target sequence, and thus avoid off-target binding.

[0045] Selective hybridization typically occurs when two nucleic acid sequences are substantially complementary (at least about 75% complementary over a length of at least 14-25 nucleotides, preferably at least about 85% complementary, more preferably at least about 90% complementary), and as a result, it is expected that a certain degree of mismatch will be tolerated.

[0046] Those skilled in the art will be able to employ suitable conditions of the desired stringency for selective hybridization, taking into account factors such as oligonucleotide length and base composition, temperature, and the like.

[0047] Suitable selective hybridization conditions for oligonucleotides of approximately 17-35 bases include hybridization in 6×SSC at 42° C. for 1 hour, followed by washing in 6×SSC at a series of increasing temperatures from 42° C. to 65° C. For example, washing may be performed using 6×SSC at 42° C. for 30 minutes, followed by washing using 6×SSC at 50° C. for 45 minutes, followed by washing using 2×SSC at 65° C. for 45 minutes. When performing a PCR reaction, the conditions for selective hybridization may include reacting the target nucleic acid with the probe / primer in a suitable buffer at about 50-60° C. for 5-10 minutes.

[0048] The degree of stringency of the wash can be varied by altering the temperature, pH, ionic strength, divalent cation concentration, volume and duration of the wash. For example, the stringency of hybridization may be varied by performing the hybridization at various temperatures below the melting temperatures of the primers or probes. For oligonucleotide probes 14-70 nucleotides in length, the melting temperature (Tm) (in degrees Celsius) may be calculated using the formula: Tm=81.5+16.6(log[Na+])+0.41(G+C moieties)-(600 / N), where N is the length of the oligonucleotide.

[0049] Other suitable conditions and protocols can be found in Molecular Cloning: A Laboratory Manual; thEdition, Green & Sambrook (2012) Cold Spring Harbor Laboratory Press NY; and Current Protocols in Molecular Biology, Ausubel et al. eds. John Wiley & Sons (2003).

[0050] As used herein, a "subject" includes a vertebrate, a mammal, a domestic animal or, preferably, a human.

[0051] A "subject," "individual," or "patient," as used herein, includes any animal that exhibits symptoms of a condition that can be detected or identified by the compositions contemplated herein. Suitable subjects include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals (e.g., horses, cows, sheep, pigs), and domestic animals or pets (e.g., cats or dogs). In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human primate, and in certain embodiments, the subject is a human.

[0052] An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of the disease, disorder, and / or condition. As referred to herein, an individual suffering from a disease (e.g., cancer) is also an individual who has the disease (e.g., cancer) or an individual in need of treatment for the disease (e.g., cancer).

[0053] As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a substance (e.g., a PRMT5 inhibitor) that partially or completely alleviates; improves; relieves; inhibits; delays the onset of; reduces the severity of; and / or reduces the frequency, occurrence or severity of one or more symptoms, characteristics and / or causes of a particular disease, disorder and / or condition (e.g., cancer). Such treatment may be treatment of subjects who do not exhibit symptoms of the associated disease, disorder and / or condition and / or subjects who exhibit only early signs of the disease, disorder and / or condition. Alternatively or in addition, such treatment may be treatment of subjects who exhibit one or more established signs of the associated disease, disorder and / or condition. In some embodiments, treatment may be treatment of subjects who have been diagnosed as suffering from the associated disease, disorder and / or condition. Treatment may be part of a "method of treatment" that may include diagnosis or selection of the patient / individual and therapeutic intervention. Patient selection may include testing the patient for suitability for being treated by therapeutic intervention, which may include testing to determine whether the patient's cancer has a relevant defect in protein or coding nucleic acid.In the context of the present invention, this may be testing to see whether the cancer contains cells that express higher than normal levels of total E2F1, and / or cells that express lower than normal levels of pRb, and / or cells that contain one or more mutations in PRB1 gene that result in reduced expression (including no expression) of pRb in cancer cells.Preferably, such testing is carried out in vitro on a biological sample that contains cancer cells from a subject that has cancer or is suspected of having cancer.

[0054] <Diagnostic method> The present invention arises from the recognition that the cancer cell targeting effect of PRMT5 inhibitors is significantly increased in cancers that express higher than normal levels of E2F1, particularly in cancers that have higher than normal levels of total E2F1, and in addition, the cancer cell targeting effect of PRMT5 inhibitors is significantly increased in cancers that express lower than normal levels of pRb (including no expression).Therefore, cancer cells with the phenotype of high total EF21 expression and / or low pRb expression are most suitable for treatment with PRMT5 inhibitors.Therefore, this discovery provides an opportunity to select patients who are most suitable for effective anti-cancer treatment with PRMT5 inhibitors.

[0055] Thus, according to a first aspect of the present invention there is provided a method of selecting a treatment for a patient with cancer comprising the steps of: (i) determining the expression level of biomarkers (a) pRb and / or (b) total E2F1 in a cancer cell-containing biological sample from a patient; (ii) comparing the expression levels in (i) with a reference value for each biomarker; (iii) if the patient's cancer cells exhibit decreased expression of pRb and / or increased expression of total E2F1 compared to baseline, the patient is selected for treatment with a PRMT5 inhibitor; A method is provided.

[0056] The expression level of a biomarker such as pRb can be determined by quantifying the amount of protein in a sample or the amount of the transcript that codes for the protein.In humans, retinoblastoma protein (protein name is abbreviated as pRb, gene name is abbreviated as Rb, RB or RB1) is encoded by the RB1 gene located at 13q14.1-q14.2 on chromosome 13.The reference sequence for the protein is disclosed by reference sequence NP_000312 in NCBI.The reference sequence for the RB1 gene is disclosed by reference sequence CCDS31973.1 in NCBI.The reference sequence is also available from G!Ensembl as ENSG00000139687 and from UniProtKB as reference P06400.

[0057] The RB1 nucleotide sequence is disclosed in SEQ ID NO:1.

[0058] The translated protein (pRb) sequence is disclosed in SEQ ID NO:2.

[0059] In humans, E2F transcription factor 1 (E2F1) is located on chromosome 20. The gene maps from 32,263,283 to 32,274,191 in GRCh37.

[0060] The reference sequence for this protein is disclosed in NCBI under reference sequence NP_005216-1. The reference sequence for the E2F1 gene is disclosed in NCBI under reference sequence CCDS13224.1. The reference sequence is also available from G!Ensembl under the name ENSG00000101412.

[0061] The E2F1 nucleotide sequence is disclosed in SEQ ID NO:3.

[0062] The translated protein sequence is disclosed in SEQ ID NO:4.

[0063] These sequences can be used to design nucleic acid probes or primers capable of binding to the transcripts, or antibodies capable of binding to the proteins, suitable for use in the invention disclosed herein.

[0064] A comparison of the expression level of the biomarker (e.g., protein or transcript amount) in the cancer cell sample must be made to assess whether the level increases, decreases, or remains the same. Such a comparison can be made against a reference value representing a wild-type or normal biomarker level. Such a reference level can be determined by assaying a wild-type cell-containing biological sample in parallel with a cancer cell-containing biological sample from a patient and comparing the expression level of the biomarker in each sample. Preferably, the two samples contain approximately the same number and type of cells to ensure comparability. Thus, if the cancer cell sample is a breast tissue sample, the comparison sample can be a matched non-cancerous breast tissue sample from the same patient or a different subject. More suitably, the typical expression level of the biomarker in the matched non-cancerous tissue has been previously determined from the analysis of many samples from non-cancerous tissue (e.g., from healthy subjects). In one approach, the average of such wild-type levels can be used as the reference value. In another approach, a threshold value can be taken from the wild-type level (e.g., the 5th percentile or the 95th percentile). Thus, for example, when it is desired to detect an increase in the expression level of a biomarker in a test sample, the value representing the 95% highest value may be taken as the reference value. Alternatively, when it is desired to detect a decrease in the expression level of a biomarker in a test sample, the value representing the 5% highest value may be taken as the reference value. It is understood that the 5% and 95% values ​​used herein are merely exemplary. The actual reference value for each biomarker will be established from a properly controlled clinical study.

[0065] Mutations in the RB1 gene can result in reduced expression of pRb compared to normal levels (Derenzini et al., 2008; Benedict et al., 1999; Bhateja et al., 2019; Lacombe et al., 2021; Xing et al., 1999). Thus, a patient's cancer can be determined to be a cancer with reduced expression levels of pRb by detecting for one or more mutations in the pRB gene that cause reduced expression.

[0066] Thus, according to a second aspect of the present invention, there is provided a method of selecting a treatment for a patient having cancer, comprising the step of determining whether the RB1 gene in the cancer cells contains one or more mutations that result in reduced expression (including no expression) of pRb, and if the cancer cells contain one or more mutations in the RB1 gene that result in reduced expression (including no expression) of pRb in the cancer cells, the patient is selected for treatment with a PRMT5 inhibitor.

[0067] Suitably, the analysis is carried out in vitro (including ex vivo) on a sample of cancer cells previously isolated from a patient.

[0068] Suitably, the mutation is selected from E137X, R251X, R255X, R320X, R358X, R445X, R455X, R467X, R552X, R556X, R579X, R787X, R661W, C712R.

[0069] The mutation identifier follows the established convention of identifying the amino acid in the wild-type protein, identifying the position of that amino acid, and identifying the amino acid that is substituted in the mutant. Thus, C712R identifies an arginine (R) substitution for a cysteine ​​(C) at position 712. X refers to the substitution for any amino acid.

[0070] Those skilled in the art can easily devise probes or primers capable of specifically identifying the presence of nucleic acids encoding the above amino acid substitution mutations.

[0071] The presence of mutation in RB1 gene can be determined using any number of well-established techniques, including nucleic acid sequencing, or amplification techniques (e.g., polymerase chain reaction) using one or more primers that act to produce an extension product only when a specific base mutation is present (e.g., allele-specific amplification), or one or more probes that can selectively distinguish nucleic acids that have or do not have one or more mutations (e.g., allele-specific hybridization).Preferably, the presence or absence of mutation in RB1 gene is determined by nucleic acid sequencing.

[0072] <Sample> The level of a biomarker (eg, E2F1 or pRb), or the presence of one or more mutations in the RB1 gene, can be determined from any suitable cancer cell-containing sample (cancer sample) of the patient.

[0073] Those skilled in the art will understand that there are many suitable examples of cancer cell samples that can be used.Suitable biological samples can be tissue samples, such as samples from biopsy or surgical resection, or biological fluid samples that contain tumor cells, such as blood, plasma, serum, sputum, saliva, pleural fluid, ascites, urine, etc.Preferably, the sample is fresh, frozen (e.g., fresh frozen), or paraffin-embedded and fixed (e.g., formalin-fixed).

[0074] In certain embodiments, the cancer sample has previously been isolated from the patient, optionally as a solid or liquid biopsy sample or during surgery. In one embodiment, the sample isolation is part of a diagnostic method.

[0075] Suitably, the cancer sample is isolated during surgical tumour resection or from a solid or liquid biopsy, such as fine needle aspiration, core needle biopsy or liquid cytology (eg a blood or peritoneal fluid sample).

[0076] In certain embodiments, the cancer sample is fresh, frozen, or paraffin-embedded and fixed.

[0077] Determination of protein levels The expression level of each biomarker can be determined based on the amount of protein present in the sample.

[0078] In one embodiment of the present invention, biomarker expression levels may be determined at the protein level. Such methods are well known in the art and include, for example, any immunohistochemistry (IHC)-based, antibody (including autoantibodies against proteins), mass spectrometry-based, and imaging (including the use of labeled ligands)-based methods known in the art and recognized as suitable for detection of proteins.

[0079] Normalization to a reference protein can then be performed to facilitate quantification of the biomarker. The normalized value can be compared to the wild-type / normal cell biomarker expression level or threshold to determine whether the cancer expresses the measured biomarker more or less than the wild-type, and / or classify the cancer as more or less likely to respond well to a PRMT5 inhibitor.

[0080] In certain embodiments, the expression level for each biomarker (eg, E2F1 or pRb) is determined based on the amount of biomarker protein detected.

[0081] In one embodiment, detection is by immunoassay using one or more antibodies specific for one or more epitopes of the biomarker protein in the cell sample of interest. Any biological material can be used for detection / quantification of the biomarker protein.

[0082] Biomarker proteins can be detected in any suitable manner, but are typically detected by contacting a sample from a patient (e.g., a sample containing cancer cells) with an antibody that binds to the biomarker protein, and then detecting the presence or absence of a reaction product. For example, by using labeled antibodies against cell surface markers, followed by fluorescence-activated cell sorting (FACS). Such antibodies are preferably labeled to allow for their easy detection after binding to the gene product. Detection methods suitable for use in the practice of the present invention include, but are not limited to, immunohistochemistry of cell-containing samples or tissues, enzyme-linked immunosorbent assays (ELISAs), including antibody sandwich assays of cell-containing tissues or blood samples, mass spectrometry, and immuno-PCR.

[0083] The antibody that can be used herein can be monoclonal, polyclonal, chimeric, or a fragment thereof. Preferably, the antibody is a monoclonal antibody. Preferably, the step of detecting the reaction product can be carried out by any suitable immunoassay.

[0084] Antibodies against known proteins, including labeled antibodies, are often commercially available.However, antibodies that can specifically bind to one of biomarkers (e.g., E2F1 or pRb) can be prepared using well-established protocols.The general techniques for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999;Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984;and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, which are incorporated herein by reference.

[0085] Full length / intact antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD and any subclass thereof, as well as fragments thereof (e.g., Fab or F(ab')2) or engineered variants thereof (e.g., sFv). Such moieties are collectively referred to as antigen-binding moieties. The antigen-binding moieties are optionally conjugated with a detectable label.

[0086] Techniques for detecting antibody binding through the use of detectable labels are well known in the art.For example, antibody binding can be detected through the use of chemical reagents that generate detectable signals that correspond to the level of antibody binding and thus the level of biomarker protein expression.In some embodiments, the detection antibody is linked to an enzyme, particularly an enzyme that catalyzes the deposition of chromogen at antigen-antibody binding sites.Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP) and alkaline phosphatase (AP).Commercially available antibody detection systems can also be used to carry out the present invention.As used herein, antibody binding also encompasses binding by antigen-binding moieties.

[0087] Although antibodies / antigen-binding moieties are exemplified herein for use in the present invention due to their extensive characterization, any other suitable agent that specifically binds to a biomarker (e.g., peptides, aptamers, or small organic molecules) may be used instead of antibodies in some cases. For example, aptamers that specifically bind to a selected biomarker may be used. Aptamers are nucleic acid-based molecules that bind to specific ligands. Methods for producing aptamers with specific binding specificities are known in the art.

[0088] As described elsewhere, the sample from the subject is typically a solid tissue sample, e.g., a biopsy, as described above, but may also be a cancer cell-containing biological fluid, e.g., a blood or serum sample. The sample may be in the form of a tissue specimen from the patient, and the specimen is suitable for immunohistochemistry in various formats, such as paraffin-embedded tissue, frozen sections of tissue, and isolated fresh tissue. Although immunodetection methods are antibody-based, there are many additional techniques that allow for sensitive determination of antibody binding with respect to tissue. Those skilled in the art are familiar with various immunohistochemistry methods.

[0089] The immunoassays performed according to the present invention may be homogeneous or non-homogeneous. In homogeneous assays, the immunological reaction usually involves a specific antibody (e.g., an anti-biomarker protein antibody), a labeled analyte, and a sample of interest. The signal resulting from the label is modified, directly or indirectly, upon binding of the antibody to the labeled analyte. Both the immunological reaction and the detection of their extent are carried out in a homogeneous solution. Immunochemical labels that can be used include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, or coenzymes.

[0090] In a non-homogeneous assay technique, the reagents are usually a sample, an antibody, and a means for producing a detectable signal. The sample described above can be used. The antibody is generally immobilized on a support such as a bead, a plate or a slide, and is contacted with a specimen suspected of containing an antigen in a liquid phase. The support is then separated from the liquid phase, and either the support phase or the liquid phase is examined for a detectable signal using a means for producing such a signal. The signal is related to the presence of the analyte in the sample. The means for producing a detectable signal include the use of a radioactive label, a fluorescent label, or an enzyme label.

[0091] For example, if the protein (or polypeptide) to be detected contains a second binding site, the antibody that binds to that site may be conjugated to a detectable group and added to the liquid-phase reaction solution before the separation step. The presence of the detectable group on the solid support indicates the presence of the antigen in the test sample. Examples of suitable immunoassays are radioimmunoassay, immunofluorescence, chemiluminescence, electrochemiluminescence or enzyme-linked immunoassay.

[0092] Those of skill in the art will be familiar with the many specific immunoassay formats and variations thereof that may be useful in carrying out the methods disclosed herein. See generally, E. Maggio, Enzyme-immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.), and also U.S. Pat. No. 4,727,022 to Skold et al., entitled "Methods for Modulating Ligand-Receptor Interactions and their Application," U.S. Pat. No. 4,659,678 to Forrest et al., entitled "Immunoassay of Antigens," U.S. Pat. No. 4,376,110 to David et al., entitled "Immunometric Assays Using Monoclonal Antibodies," U.S. Pat. No. 4,275,149 to Litman et al., entitled "Macromolecular Environment Control in Specific Receptor Assays," and U.S. Pat. No. 4,233,402 to Maggio et al., entitled "Reagents and Method Employing No. 4,230,767 to Boguslaski et al., entitled "Heterogenous Specific Binding Assay Employing a Coenzyme as Label."

[0093] The antibody / antigen-binding portion can be conjugated to a solid support suitable for diagnostic assays (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) according to known techniques, such as passive binding.

[0094] The antibody / antigen binding portions described herein can similarly be conjugated to detectable groups such as radiolabels (e.g., 35S, 125I, 131I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) according to known techniques.

[0095] Alternative methods for detecting protein biomarkers in a sample include high performance liquid chromatography (HPLC) and other high-throughput techniques.

[0096] In addition, the identification and quantification of one or more biomarkers can be performed using mass spectrometry. One particular example of mass spectrometry that can be useful is tandem mass spectrometry, and another example is high mass accuracy / high mass resolution mass spectrometry (e.g., Orbitrap™, Thermo Scientific).

[0097] For example, tandem mass spectrometry can be used for the quantitative analysis of peptides in biological samples due to its high sensitivity and high specificity. High mass accuracy / high mass resolution mass spectrometers (e.g., Orbitrap™, Thermo Scientific) can also be utilized for analysis.

[0098] <Determination of RNA transcript levels> RNA transcript expression levels can be used as a surrogate measurement of the level of protein in a sample. RNA transcript expression levels can be determined either at the RNA level (i.e., mRNA or non-coding RNA (ncRNA)) (e.g., miRNA, tRNA, rRNA, snoRNA, siRNA and piRNA) or at the protein level. Measuring gene expression at the mRNA level includes measuring the level of cDNA corresponding to the mRNA. Those skilled in the art are familiar with a variety of techniques for determining the status of genes or proteins in tissue or cell samples, including, but not limited to, microarray analysis (e.g., for assaying mRNA or microRNA expression, copy number, etc.), real-time PCR (RTPCR), quantitative real-time PCR (qRT-PCR, e.g., TaqMan™), digital PCR (dPCR), microarray, high-throughput sequencing (also known as next-generation sequencing, e.g., RNA-seq), sequential analysis of gene expression (SAGE) and digital gene expression (DGE), etc.

[0099] In certain embodiments, the expression level of each biomarker gene may be determined relative to various features of the gene's expression product, including exons, introns, protein epitopes, and protein activity.

[0100] In certain embodiments, the RNA transcript expression level for each gene to be measured is quantitatively determined.Quantitative measurement typically involves the parallel measurement of the expression level of one or more standard or housekeeping genes to determine the normalized expression level of test RNA transcript.This is to ensure that approximately the same amount of test sample is compared with the same amount of control sample and / or reference value or threshold value.

[0101] In a particular embodiment, the determination of the transcript expression level according to the first or second aspect of the invention is carried out using RT-PCR.

[0102] In certain embodiments, the RT-PCR is quantitative reverse transcription polymerase chain reaction (RT-qPCR).

[0103] Messenger RNA isolation, purification, primer extension and amplification can be performed according to methods available in the art (see, for example, Godfrey et al. J. Molec. Diagnostics 2: 84-91 (2000); Specht et al., Am. J. Pathol. 158: 419-29 (2001)). TaqMan® RT-PCR can be performed using commercially available equipment such as, for example, the QuantStudio™ 7 Real-Time PCR System (Thermo Fisher Scientific) or the Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In a preferred embodiment, the 5' nuclease procedure is performed on a quantitative real-time PCR device such as the QuantStudio™ 5 Real-Time PCR System. The system consists of a thermocycler, a light-emitting diode (LED) and light wavelength filters, a Complementary Metal-Oxide Semiconductor (CMOS) camera and a computer. This system amplifies samples in a 96-well format on a thermocycler. RT-PCR can be performed in triplicate wells with 2ng equivalent RNA input per 10μl reaction volume. During amplification, the fluorescent signal induced by the LED is collected in real time, for example, through a fiber optic cable for every well, and detected in the CMOS. The system includes software for running the instrument and for analyzing the data.

[0104] In certain embodiments, RT-qPCR is performed using primers capable of selectively hybridizing to target gene transcripts in the panel of genes.

[0105] In certain embodiments, the level of the RNA transcript for each gene is normalized, such as by reference to the transcript level of at least one reference gene.

[0106] In certain embodiments, RT-qPCR is performed on total RNA extracted from one or more slices or sections of the cancer sample.

[0107] In certain embodiments, RNA transcript expression levels are measured using RT-PCR to determine cycle threshold (Ct) levels. Ct levels refer to the number of rounds or cycles of PCR carried out until a certain threshold of amplification products is produced. In real-time PCR assays, positive reactions are detected by the accumulation of a signal, typically a fluorescent signal. Ct is defined as the number of cycles required for the signal to exceed a threshold, typically background level. Ct is sometimes referred to as Cq (quantification cycle). Ct levels are inversely proportional to the amount of target nucleic acid in a sample.

[0108] Current PCR instruments are capable of collecting fluorescence data during each cycle and calculating Ct values.

[0109] The measured Ct level can then be normalized using the Ct of the reference gene to obtain delta Ct (dCt). Delta Ct is the normalized Ct level that corresponds to the difference between the Ct of the test gene and the Ct of the reference gene (or the average of the reference genes if more than one reference gene is used in the experiment). Preferably, gene expression measurements can be normalized to the average value of one or more (e.g., 2, 3, 4, 5 or more) reference genes.

[0110] The level of protein in tumor sample can be used as a proxy measurement of RNA transcript expression level.The level of protein in tumor sample can be determined by any technique known in the art, for example, HPLC, mass spectrometry, or using antibody specific for selected protein (for example, IHC, ELISA, etc.).Then, normalization to reference protein can be performed in the same manner as for RNA detection, or as a ratio to reference, or by other standard methods.

[0111] Real-time PCR is compatible with both quantitative competitive PCR, in which the internal competitor for each target sequence is used for normalization, and quantitative comparative PCR, which uses the normalization gene contained in the sample or the housekeeping gene for RT-PCR.For further details, see, for example, Held et al., Genome Research 6:986-994 (1996).

[0112] Other suitable techniques that can be used include digital PCR or sequential analysis of gene expression (SAGE).For further details, see, for example, Velculescu et al., Science 270:484-487 (1995); and Velculescu et al., Cell 88:243-51 (1997).

[0113] <Gene expression analysis by nucleic acid sequencing> Nucleic acid sequencing techniques can also be used to detect gene expression levels. The premise is that the number of times a cDNA sequence is detected in a sample is directly related to the relative expression of the mRNA corresponding to that sequence. These methods are sometimes referred to by the term Digital Gene Expression (DGE), which reflects the discrete numerical nature of the data obtained. Early methods that applied this principle were Serial Analysis of Gene Expression (SAGE) and Massively Parallel Signature Sequencing (MPSS). See, for example, S. Brenner, et al., Nature Biotechnology 18(6):630-634 (2000). More recently, the advent of "next-generation" sequencing technologies has made DGE simpler, more high-throughput, and more affordable. One such example is RNA sequencing ("RNA-seq"), where cDNA molecules are synthesized from RNA, sequenced in high-throughput, and aligned to a reference standard. RNA expression can then be determined in a highly sensitive manner by the number of aligned reads. Furthermore, individual transcript splice variants can be identified and quantified, and genetic mutations or variants, such as single nucleotide polymorphisms (SNPs), can be determined.

[0114] <Data Analysis> In certain embodiments, the expression level of a biomarker can be compared to the expression level detected in a control cell, which can be obtained from a non-cancerous tissue from the same or different individual. Suitable controls include non-cancerous cells from the same tissue or lineage. Comparisons can be performed on test samples and reference samples measured at the same time or at separate times in time. An example of the latter is the use of a sequence database to assemble aggregated expression information, such as information about the expression level of a biomarker.

[0115] In some embodiments, the expression of one or more reference (sometimes called "housekeeping") genes or proteins is also obtained for use in normalizing the expression of the test genes / proteins. As used herein, "reference gene or protein" refers to a gene or protein whose expression is used to calibrate or normalize the measured expression of the test protein / gene of interest. Normalization ensures accurate comparison of the expression of the test biomarker between different samples. For this purpose, reference genes / proteins known in the art can be used. Examples of reference genes / proteins to which biomarker expression levels can be normalized include, but are not limited to, GAPDH and / or actin.

[0116] The increase or decrease in expression of the biomarkers disclosed herein can be determined based on percent or fold change relative to expression in normal cells, reference cells, or can be normalized to one or more reference or housekeeping biomarkers.The increase can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or 200% increase relative to expression levels in normal cells.Alternatively, the fold increase can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10-fold increase relative to expression levels in normal cells. The decrease can be 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% decrease with respect to the expression level in normal cells. For example, a 2-fold increase or decrease is a useful measurement for determining whether the expression level is low or high. Advantageously, the threshold level of expression for predicting or recruiting (e.g., whether a patient is likely to respond to treatment with a PRMT5 inhibitor) based on the method of the present invention can be empirically determined using clinical samples.

[0117] <Medical Uses and Treatment Methods> According to a fourth aspect of the present invention there is provided a PRMT5 inhibitor or a pharmaceutical composition comprising a PRMT5 inhibitor for use in treating a patient having a cancer in which the cancer cells express higher than normal levels of the E2F1 biomarker and / or express reduced levels of the pRb biomarker compared to normal.

[0118] In a variant of the fourth aspect of the present invention, there is provided a method of treating a patient having a cancer in which the cancer cells express higher than normal levels of the E2F1 biomarker and / or express reduced levels of the pRb biomarker compared to normal, comprising the step of administering to the patient an effective amount of a PRMT5 inhibitor or a pharmaceutical composition comprising a PRMT5 inhibitor.

[0119] In a variation of the fourth aspect of the invention, there is provided a method of treating a patient with cancer, comprising the steps of: (i) determining the expression level of the total E2F1 biomarker and / or the pRb biomarker in a cancer cell sample from the patient; (ii) comparing the level determined in step (i) with the expression level in a normal cell; (iii) if the patient's cancer cells express a higher than normal level of the E2F1 biomarker and / or express a reduced level of the pRb biomarker compared to normal, the patient is administered an effective amount of a PRMT5 inhibitor or a pharmaceutical composition comprising a PRMT5 inhibitor. A method is provided.

[0120] In a variant of the fourth aspect of the invention, there is provided the use of a PRMT5 inhibitor in the manufacture of a medicament for use in treating a patient having a cancer whose cancer cells express higher than normal levels of the E2F1 biomarker and / or express reduced levels of the pRb biomarker compared to normal.

[0121] Suitably, the patient has been identified according to the first or second aspect of the invention, whose cancer cells express higher than normal levels of the E2F1 biomarker and / or reduced levels of the pRb biomarker compared to normal.

[0122] In certain embodiments, the expression level of the E2F1 biomarker is the total E2F1 protein level.

[0123] In certain embodiments, the expression level of the E2F1 biomarker is the E2F1 transcript level.

[0124] In certain embodiments, the expression level of the pRb biomarker is a pRb protein level.

[0125] In a particular embodiment, the expression level of the pRb biomarker is the RB1 transcript level.

[0126] According to a fifth aspect of the invention there is provided a PRMT5 inhibitor for use in the treatment of a pRb deficient cancer or for use in the treatment of a cancer whose cells express reduced levels of the pRb biomarker compared to normal.

[0127] In a variant of the fifth aspect of the present invention, there is provided a method of treating a patient having a pRb deficient cancer or whose cancer cells have reduced levels of the pRb biomarker compared to normal, comprising the step of administering to the patient an effective amount of a PRMT5 inhibitor or a pharmaceutical composition comprising a PRMT5 inhibitor.

[0128] In a variant of the fifth aspect of the invention, there is provided the use of a PRMT5 inhibitor in the manufacture of a medicament for use in treating a patient having a pRb deficient cancer or whose cancer cells have reduced levels of the pRb biomarker compared to normal.

[0129] Suitably, the patient, whose cancer cells express reduced levels of the pRb biomarker compared to normal, has been identified according to the first or second aspect of the invention.

[0130] In certain embodiments, the expression level of the pRb biomarker is a pRb protein level.

[0131] In a particular embodiment, the expression level of the pRb biomarker is the RB1 protein level.

[0132] In certain embodiments, patients whose cancer cells express reduced levels of the pRb biomarker compared to normal have been identified by their cancer cells containing one or more mutations in the RB1 gene that result in reduced expression (including no expression) of pRb.

[0133] In certain embodiments, the one or more mutations in the RB1 gene that result in reduced expression (including no expression) of pRb are selected from E137X, R251X, R255X, R320X, R358X, R445X, R455X, R467X, R552X, R556X, R579X, R787X, R661W, and C712R.

[0134] In a variation of the fourth aspect of the invention, there is provided a method of treating a patient with cancer, comprising the steps of: (i) determining whether the RB1 gene in cancer cells in a cancer cell sample from a patient contains one or more mutations that result in reduced expression of pRb; (ii) if the RB1 gene in the cancer cells contains one or more mutations that result in reduced expression of pRb, the patient is administered an effective amount of a PRMT5 inhibitor or a pharmaceutical composition containing a PRMT5 inhibitor. A method is provided.

[0135] In certain embodiments of the third or fourth aspect of the present invention, the PRMT5 inhibitor is for use in a method of treating cancer in which the cells express a higher than normal level of the E2F1 biomarker and a reduced level of the pRb biomarker compared to normal. As described above, a reduced level of the pRb biomarker compared to normal can be determined by detecting the presence of one or more mutations in the RB1 gene that result in a reduction in the expression of pRb.

[0136] Any therapeutic agent capable of inhibiting PRMT5 can be used in the fourth or fifth aspect of the present invention. In certain embodiments, the PRMT5 inhibitor can be selected from the group consisting of an antibody, an RNA interference molecule (e.g., microRNA / miRNA, small interfering RNA / siRNA, or small hairpin RNA / shRNA), an antisense oligonucleotide (ASO), or a small molecule compound.

[0137] Preferably, the PRMT5 inhibitor for use in the third or fourth aspect of the present invention relates to a pharmaceutical composition comprising the PRMT5 inhibitor and at least one pharmaceutically acceptable component.

[0138] <PRMT5 inhibitor> Any therapeutic agent capable of inhibiting PRMT5 can be used in the fourth or fifth aspect of the present invention. In certain embodiments, the PRMT5 inhibitor can be selected from the group consisting of an antibody, an RNA interference molecule (e.g., microRNA / miRNA, small interfering RNA / siRNA, or small hairpin RNA / shRNA), an antisense oligonucleotide (ASO), or a small molecule compound.

[0139] In certain embodiments of any of the aspects disclosed herein, the PRMT5 inhibitor is a small molecule compound or a macromolecule.

[0140] Preferably, the PRMT5 inhibitor is selected from the group consisting of an antibody, a peptide, a nucleic acid, a small molecule compound, an RNA inhibitory molecule (RNAi) and an antisense oligonucleotide (ASO).

[0141] In a particular embodiment, the PRMT5 inhibitor or pharmaceutical composition thereof for use according to the fourth or fifth aspect of the invention causes a reduction in the functional activity of PRMT5 or the expression level of PRMT5.

[0142] <Nucleic acid inhibitors> In certain embodiments, the PRMT5 inhibitor for use in the present invention is a nucleic acid-based therapeutic agent comprising nucleic acid or nucleotide.For example, said nucleic acid therapeutic agent can be or comprises dsRNA molecule, RNAi molecule, miRNA molecule, ribozyme, shRNA molecule, antisense oligonucleotide (ASO), guide RNA (gRNA) or siRNA molecule.

[0143] RNAi and ASO molecules are particularly suitable for inhibiting the expression of PRMT5. The use of these techniques to downregulate gene expression is now well established in the art.

[0144] In certain embodiments, the PRMT5 inhibitor is an RNAi.

[0145] In certain embodiments, the PRMT5 inhibitor is an ASO.

[0146] Also, the PRMT5 inhibitor for use in the present invention can be a nucleic acid-based molecule that can inhibit PRMT5 mRNA.There are many different kinds of nucleic acid-based molecules that can inhibit the translation of mRNA and / or reduce the stability of RNA.Such RNA inhibitor is preferably PRMT5 mRNA-specific RNAi molecule, PRMT5 mRNA-specific shRNA molecule, or PRMT5 mRNA-specific antisense oligonucleotide (AON).

[0147] Thus, according to another embodiment, the PRMT5 inhibitor is or comprises a nucleic acid molecule capable of inhibiting the mRNA of PRMT5.

[0148] <Antibody> Also, the PRMT5 inhibitor can be a large biomolecule, for example, an antibody or an antibody fragment. In certain embodiments, the PRMT5 inhibitor is a monoclonal antibody. In certain embodiments, the PRMT5 inhibitor is a monoclonal antibody fragment. In certain embodiments, the PRMT5 inhibitor is a polyclonal antibody. In certain embodiments, the PRMT5 inhibitor is an intracellular antibody (intrabody).

[0149] An antibody is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable domain of the immunoglobulin molecule.

[0150] An "intact antibody" typically refers to a Y-shaped tetrameric protein that contains two heavy (H) and two light (L) polypeptide chains linked by covalent disulfide bonds and non-covalent interactions. Each light chain consists of one variable domain (VL) and one constant domain (CL). Each heavy chain contains one variable domain (VH) and a constant region, which contains three domains called CH1, CH2 and CH3 for IgG, IgA and IgD antibodies (IgM and IgE have a fourth domain, CH4). In the IgG, IgA and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a proline- and cysteine-rich segment of variable length (from about 10 to about 60 amino acids in the various IgG subclasses). The variable domains of both the light and heavy chains are joined to the constant domains by a "J" region of about 12 or more amino acids, and the heavy chains also have a "D" region of about 10 additional amino acids. Each class of antibody further comprises inter- and intrachain disulfide bonds formed by paired cysteine ​​residues. The heavy chain variable region (YH) and the light chain variable region (YL) can each be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each YH and YL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of complement (C1q) of the classical complement system.

[0151] As used herein, the term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring intact antibodies, e.g., polyclonal, multiclonal or monoclonal antibodies, as well as chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, polyvalent antibodies, anti-idiotypic antibodies and synthetic antibodies, but also includes, unless otherwise specified, any antigen-binding portion thereof that competes with the intact antibody for specific binding, fusion proteins comprising an antigen-binding portion, and any other modified configuration of an immunoglobulin molecule that comprises an antigen recognition site.

[0152] It has been shown that the antigen-binding function of an antibody can be performed by a portion of a full-length antibody. An antigen-binding portion of an antibody (also called an "antigen-binding fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen (e.g., PRMT5) bound by the whole antibody.

[0153] Antigen-binding moieties include, for example, Fab, Fab', F(ab')2, F(ab') fragments, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), moieties containing complementarity determining regions (CDRs), single chain variable fragment antibodies (e.g., scFv, scFvFc and bis-scFv), minibodies, maxibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to a polypeptide. Depending on the antibody amino acid sequence of the constant region of the heavy chain, immunoglobulins can be assigned to different classes. There are five major classes (i.e., isotypes) of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (subtypes), e.g., IgG1, lgG2, lgG3, lgG4, IgA1, and lgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Unless otherwise dictated by contextual constraints, the term further includes all classes and subclasses of antibodies. The heavy chain constant domains corresponding to different classes of antibodies are typically designated by the corresponding lowercase Greek letters α, δ, ε, γ and μ, respectively. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0154] Although the two domains of the Fv portion, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker that allows them to be produced using recombinant methods as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv)). See, for example, Bird et al. Science 242:423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, allowing these domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993; Poljak et al., Structure. 2:1121 -1123, 1994).

[0155] The antibody may be murine, rat, human or of any other origin, including chimeric or humanized antibodies. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a human or humanized antibody. A non-human antibody may be humanized by recombinant methods to reduce its immunogenicity in humans.

[0156] The term "monoclonal antibody" ("mAb") refers to antibody molecules of single molecular composition, i.e., a non-natural preparation of antibody molecules essentially identical in primary sequence. Mabs are highly specific, being directed to a single antigenic site / epitope. A mAb is an example of an isolated antibody. mAbs may be produced by hybridoma, recombinant, transgenic, or other techniques known to those of skill in the art. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present disclosure may be produced by the hybridoma method first described by Kohler and Milstein (Nature 256:495, 1975), or may be produced by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., (Nature 348:552-554, 1990), for example.

[0157] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. A human antibody may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously. This definition of a human antibody specifically excludes humanized antibodies that include non-human antigen-binding residues.

[0158] As used herein, a "humanized antibody" refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced with the corresponding amino acids from a human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's CDRs are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat or rabbit that has the desired specificity, affinity and capacity. A humanized antibody may contain residues that are not found in the recipient antibody or in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In one embodiment of a humanized form of Ab, some, most or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, while some, most or all of the amino acids within one or more CDR regions are left unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are acceptable if they do not abrogate the ability of the antibody to bind to a particular antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0159] "Chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, e.g., the variable region is derived from a mouse antibody and the constant region is derived from a human antibody, or vice versa. The term also encompasses antibodies that contain a V region derived from one individual of one species (e.g., a first mouse) and a constant region derived from another individual of the same species (e.g., a second mouse).

[0160] "Intrabody" refers to an antibody that is designed to be expressed intracellularly through in-frame fusion with an intracellular localization peptide sequence and can target specific target antigens present in various subcellular locations including the cytoplasm, nucleus and endoplasmic reticulum. It has been identified as a new class of therapeutic molecule (Chen et al., Human Gene Therapy. 5(5): 595-601, 1994). Intrabodies can be expressed in various forms, but the most commonly used format is the scFv due to its mall size. Antibody fragments, typically in the scFv format, are cloned into specific targeting vectors, allowing expression of the intrabody in the nucleus, cytoplasm or ER. The intrabody genes are expressed in the target cell after transfection of an expression plasmid or viral transduction of a recombinant virus. It has been found that the usual vectors, promoters and transfection systems for heterologous expression can be used to express intrabodies in the cells of interest.

[0161] Generally, the term "epitope" refers to the area or region of an antigen to which an antibody specifically binds, i.e., the area or region that is in physical contact with the antibody. Thus, the term "epitope" refers to that portion of a molecule that is recognized by an antibody at one or more of the antigen-binding regions of the antibody and capable of being bound by the antibody.

[0162] An antibody that "specifically binds" to an epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with greater affinity than it reacts or associates with alternative cells or substances. For example, an antibody that specifically binds to a PRMT5 epitope is an antibody that binds to this epitope with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other PRMT5 epitopes or non-PRMT5 epitopes. It is also understood by reading this definition that, for example, an antibody that specifically binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" does not necessarily require exclusive binding (although "specific binding" may include exclusive binding).

[0163] For example, the antibody against PRMT5 can be produced by any method known in the art.The general techniques for producing human and mouse antibodies are known in the art and / or described herein.See, for example, Harlow and Lane (1988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NJ.

[0164] In some embodiments, the antibody can be recombinantly produced and expressed using any method known in the art. In some embodiments, the antibody can be prepared and selected by phage display technology. See, for example, U.S. Patent Nos. 5,565,332, 5,580,717, 5,733,743 and 6,265,50, and Winter et al., (Annu. Rev. Immunol. 12:433-455, 1994). Alternatively, phage display technology (McCafferty et al., Nature 348:552-553, 1990) can be used to produce human antibodies and antibody portions in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

[0165] In certain embodiments, the antibody for use in the present invention is selected from a monoclonal, human, humanized, Fab, Fab', F(ab')2, F(ab'), Fd, Fv, dAb, intrabody, scFV and VHH antibody.

[0166] Antibody and nucleic acid technology molecule (eg RNAi and ASO) technologies have evolved accordingly and a person skilled in the art may be able to produce antibodies or antibody derived molecules or nucleic acid technology molecules capable of inhibiting PRMT5.

[0167] <Small molecule PRMT5 inhibitor compound> In certain embodiments, the PRMT5 inhibitor is a small molecule compound.

[0168] A "small molecule" as used herein is an organic molecule with a mass of less than about 5 kilodaltons (KDa). In some embodiments, a small molecule is less than about 3 KDa, or less than about 2 KDa, or less than about 1.5 KDa, or less than about 1 KDa. Most small molecule compounds are less than about 800 Daltons (Da). In some embodiments, a small molecule is less than about 800 Da, less than about 600 Da, less than about 500 Da, less than about 400 Da, less than about 300 Da, less than about 200 Da, or less than about 100 Da. In many cases, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule is non-polymeric. In some embodiments, small molecules contain multiple carbon-carbon bonds and may include one or more heteroatoms and / or one or more functional groups important for structural interactions (e.g., hydrogen bonding) with proteins, such as amines, carbonyls, hydroxyl or carboxyl groups, and in some embodiments at least two functional groups. Small molecules often contain one or more carbocyclic or heterocyclic structures and / or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups.

[0169] PRMT5 inhibitor compounds with distinct chemophores are known. Fereira de Freitas et al., (Molecules. 24:4492, 2019) provides an overview of some of the PRMT5 inhibitors, lists their structures, and outlines their mechanism of action.

[0170] The following table lists some of the patent publications filed by various pharmaceutical companies and others that are directed to PRMT5 inhibitors that may be used in the present invention.

[0171] [Table 1]

[0172] Compound 208 of WO2014 / 100719 (Epizyme) is GSK3326595 (pemurametostat).

[0173] The compound of Example 2 in WO2016 / 178870 (Eli Lilly) is LLY-283.

[0174] Compound 80 of WO2017 / 032840 (Janssen Pharmaceuticals) is JNJ-64619178.

[0175] See also Fereira de Freitas et al., (Molecules. 24:4492, 2019).

[0176] Other suitable PRMT5 inhibitors include (1) A compound of formula I or a salt, solvate or hydrate thereof, as disclosed in WO2018 / 167269 (Argonaut Therapeutics Limited): [ka] {In the formula, R1, R3, R4, R5 and R6 are each independently hydrogen or C 1~3 alkyl, R2 is hydrogen and R 14 is selected from X is O or NR9, where R9 is hydrogen or C 1~3 is alkyl, Y1 is a group represented by the formula A and B, [ka] where each R''' is independently selected from H and C 1~3 alkyl, Q is C or N; T is selected from fused phenyl groups and fused 5- or 6-membered heteroaryl groups, each of which is optionally selected from halo and C 1~3 substituted by one or more substituents selected from alkyl, R7 and R8, together with the nitrogen atom between them, form a 3- to 12-membered heterocycloalkyl ring, where the 3- to 12-membered heterocycloalkyl ring optionally contains one or more R 10 and / or optionally one or more C 6~12 Aryl, C 5~12 Heteroaryl, C 3~8 cycloalkyl and 3- to 12-membered heterocycloalkyl rings, where each fused C 6~12 Aryl, C 5~12 Heteroaryl, C 3~8 Cycloalkyl and 3- to 12-membered heterocycloalkyl rings optionally contain one or more R 14 is replaced by R 10 is the formula L1-L2-R 11 or L2-L1-R 11 wherein L1 is selected from the group of formula -[CR 12 R 13 ] n -, where n is an integer from 0 to 3, and R 12 and R 13 is independently selected from H and C1-C2 alkyl, where L2 is absent or O, S, SO, SO2, N(R'), C(O), C(O)O, [O(CH2) r ] s , [(CH2) r O] s is a linker selected from OC(O), CH(OR'), C(O)N(R'), N(R')C(O), N(R')C(O)N(R'), SON(R') or N(R')SO, where R' and R'' are each independently selected from hydrogen and C1-C2 alkyl, where r is 1 or 2 and s is 1-4; R 11are independently hydrogen, CN, NO2, hydroxyl, =O, halogen, C 1~6 Haloalkyl, C 1~6 Haloalkoxy, C 1~6 Alkyl, OC 1~6 Alkyl, C 3~6 Cycloalkyl, C 6~12 Aryl, C 5~12 Heteroaryl, 3-10 membered heterocycloalkyl, -C(=O)R d , -C(=O)OR d , -C(=O)NR e R d , -C(O)C(=O)R d , -NR e R d , -NR e C(=O)R d , -NR e C(=O)OR d , -NR e C(=O)NR e R d , -NR e S(=O)2R d , -NR e S(=O)2NR e R d , -OR d , -SR d , -OC(=O)R d , -OC(=O)NR e R d , -OC(=O)OR d , -S(=O)2R d , -S(=O)R d , -OS(=O)R d , -OS(=O)2R d , -OS(=O)2OR d , -S(=O)NR e R d , -OS(=O)2NR e R d and -S(=O)NR e R d where R 11 is independently 3~6 Cycloalkyl, C 6~12 Aryl, C 5~12heteroaryl and 3- to 10-membered heterocycloalkyl, each C 3~6 Cycloalkyl, C 6~12 Aryl, C 5~12 Heteroaryl and 3- to 10-membered heterocycloalkyl optionally include one or more R 14 is replaced by Each R a and R b are independently hydrogen and C 1~6 alkyl, Each R d are independently hydrogen, hydroxyl, halogen, CN, C 1~6 Haloalkyl, 3-7 membered heterocycloalkyl, C 3~6 Cycloalkyl, C 1~6 Alkyl, OC 1~6 Alkyl and C 6~11 aryl, wherein said C 1~6 Alkyl, C 6~11 Aryl, 3-7 membered heterocycloalkyl and C 3~6 Cycloalkyl is optionally substituted with hydroxyl, ═O, halogen, CN, COR a , N.R. a R b , C 1~6 Haloalkyl, C 3~6 Cycloalkyl, C 6~11 Aryl, 3-7 membered heterocycloalkyl, C 1~6 Alkyl and OC 1~6 substituted with one or more groups selected from alkyl, Each R e are independently hydrogen, hydroxyl, halogen, CN, C 1~6 Haloalkyl, C 3~6 Cycloalkyl, C 1~6 Alkyl and OC 1~6 alkyl; or R e and R d When attached to the same atom, they are treated as hydroxyl, =O, halogen, CN, COR, together with the atom to which they are attached. a , N.R. a R b , C1~6 Haloalkyl, C 3~6 Cycloalkyl, C 6~11 Aryl, 3-7 membered heterocycloalkyl, C 1~6 Alkyl and OC 1~6 forming a 3- to 7-membered heterocycloalkyl ring optionally substituted with one or more substituents selected from alkyl; R 14 are independently halo, CN, NO2, hydroxyl, =O, halogen, C 1~6 Haloalkyl, C 1~6 Haloalkoxy, C 1~6 Alkyl, OC 1~6 Alkyl, C 3~6 Cycloalkyl, C 6~12 Aryl, 5-6 membered heteroaryl, 3-7 membered heterocycloalkyl, C 1~6 Alkyl C 6~12 Aryl, -C(=O)R d , -C(=O)OR d , -C(=O)NR e R d , -C(O)C(=O)R d , -NR e R d , -NR e C(=O)R d , -NR e C(=O)OR d , -NR e C(=O)NR e R d , -NR e S(=O)2R d , -NR e S(=O)2NR e R d , -OR d , -SR d , -OC(=O)R d , -OC(=O)NR e R d , -OC(=O)OR d , -S(=O)2R d , -S(=O)R d , -OS(=O)R d , -OS(=O)2R d , -OS(=O)2OR d , -S(=O)NRe R d , -OS(=O)2NR e R d and -S(=O)NR e R d selected from (2) A compound of formula I or a salt, solvate or hydrate thereof, as disclosed in WO2018 / 167276 (Argonaut Therapeutics Limited); [ka] {In the formula, Y 1 is represented by formulas A and B, [ka] is a group selected from one of X is O, S, CH, or NR 7 is selected from X 1 is selected from C and N; Y is selected from fused aryl groups and fused heteroaryl groups, each of which is optionally selected from one or more R 11 is replaced by n is 1 and L is -(CH2) p N(R a )C(O)-, -(CH2) p C(O)N(R a )-, -(CH2) p N(R a )S(O q )-, -(CH2) p S(O q )N(R a )-, -(CH2) p N(R b )C(O)N(R b )-, -(CH2) p N(R c )C(O)O- and -(CH2) p O C (O) N (R c )-or n is 0 and L is R d (Re )NC(O)-, -R d (R e )NC(O)N(R b )-, R d (R e )NC(O)O-, R d (R e )NS(O q ) and R d (R e )N-, p is a number selected from 0, 1, 2 and 3; q is a number selected from 1 and 2; Z is one or more R 10 C optionally replaced by 6~11 Aryl, one or more R 10 Optionally replaced by (C 7~16 ) alkylaryl, one or more R 10 C optionally replaced by 3~11 Cycloalkyl, one or more R 10 Optionally replaced by (C 4~17 ) cycloalkylalkyl, one or more R 10 3 to 15 membered heterocycloalkyl optionally substituted with one or more R 10 4 to 21 membered alkylheterocycloalkyl optionally substituted with one or more R 10 and one or more R 10 selected from 6-21 membered alkylheteroaryl optionally substituted by R 1 is hydrogen, halogen, -NR e R d , OR f , and one or more R 9 C optionally replaced by 1~6 alkyl, R 2 is hydrogen, halogen, and one or more R 9 C optionally replaced by 1~6alkyl, R 3 , R 4 , R 5 and R 6 are independently selected from hydrogen, halogen, and one or more R 9 C optionally replaced by 1~6 alkyl, R 7 is hydrogen, hydroxyl, C 1~6 Alkyl, C 1~6 Haloalkyl, Phenyl and C 3~6 cycloalkyl, wherein said C 1~6 Alkyl, phenyl and C 3~6 Cycloalkyl is optionally substituted with hydroxyl, halogen, ═O, CN, COR a , N.R. a R b , C 1~6 Haloalkyl, C 3~6 Cycloalkyl, C 6~11 Aryl, 3-7 membered heterocycloalkyl, C 1~6 Alkyl and OC 1~6 substituted by one or more substituents selected from alkyl, Each R 9 are independently hydrogen, hydroxyl, halogen, CN, C 1~6 Haloalkyl, 3-7 membered heterocycloalkyl, C 3~6 Cycloalkyl, C 1~6 Alkyl, OC 1~6 alkyl and phenyl, 1~6 Alkyl, phenyl, 3-7 membered heterocycloalkyl and C 3~6 Cycloalkyl is optionally substituted with hydroxyl, ═O, halogen, CN, NR a R b , C.O.R. a , C 1~6 Haloalkyl, C 3~6 Cycloalkyl, phenyl, 3-7 membered heterocycloalkyl, C 1~6 Alkyl and OC 1~6 substituted with one or more groups selected from alkyl, Each R10 are independently hydrogen, hydroxyl, =O, halogen, CN, C 1~6 Haloalkyl, C 1~6 Haloalkoxy, C 1~6 Alkyl, OC 1~6 Alkyl, C 3~6 Cycloalkyl, phenyl, 5-6 membered heteroaryl, 3-7 membered heterocycloalkyl, -C(=O)R d , -C(=O)OR d , -C(=O)NR e R d , -C(O)C(=O)R d , -NR e R d , -NR e C(=O)R d , -NR e C(=O)OR d , -NR e C(=O)NR e R d , -NR e S(=O)2R d , -NR e S(=O)2NR e R d , -OR d , -SR d , -OC(=O)R d , -OC(=O)NR e R d , -OC(=O)OR d , -S(=O)2R d , -S(=O)R d , -OS(=O)R d , -OS(=O)2R d , -OS(=O)2OR d , -S(=O)NR e R d , -OS(=O)2NR e R d and -S(=O)NR e R d wherein C is selected from 3~6 Cycloalkyl, C 1~6 Alkyl, phenyl, 5- to 6-membered heteroaryl and 3- to 7-membered heterocycloalkyl are optionally substituted with hydroxyl, halogen, ═O, CN, C 1~6 Haloalkyl, C1~6 Haloalkoxy, C 3~6 Cycloalkyl, C 1~6 Alkyl and OC 1~6 substituted with one or more groups selected from alkyl, R 11 is hydrogen, hydroxyl, halogen, CN, NR a R b , C 1~6 Haloalkyl, 3-7 membered heterocycloalkyl, C 3~6 Cycloalkyl, C 1~6 Alkyl, OC 1~6 alkyl and phenyl, 1~6 Alkyl, phenyl, 3-7 membered heterocycloalkyl and C 3~6 Cycloalkyl is optionally substituted with hydroxyl, ═O, halogen, CN, COR a , N.R. a R b , C 1~6 Haloalkyl, C 3~6 Cycloalkyl, C 6~11 Aryl, 3-7 membered heterocycloalkyl, C 1~6 Alkyl and OC 1~6 substituted with one or more groups selected from alkyl, Each R a , R b and R c are independently hydrogen and C 1~6 alkyl, Each R d are independently hydrogen, hydroxyl, halogen, CN, C 1~6 Haloalkyl, 3-7 membered heterocycloalkyl, C 3~6 Cycloalkyl, C 1~6 Alkyl, OC 1~6 Alkyl and C 6~11 aryl, wherein said C 1~6 Alkyl, C 6~11 Aryl, 3-7 membered heterocycloalkyl and C 3~6 Cycloalkyl is optionally substituted with hydroxyl, ═O, halogen, CN, COR a , N.R. a Rb , C 1~6 Haloalkyl, C 3~6 Cycloalkyl, C 6~11 Aryl, 3-7 membered heterocycloalkyl, C 1~6 Alkyl and OC 1~6 substituted with one or more groups selected from alkyl, Each R e are independently hydrogen, hydroxyl, halogen, CN, C 1~6 Haloalkyl, C 3~6 Cycloalkyl, C 1~6 Alkyl and OC 1~6 alkyl; or R e and R d When attached to the same atom, they are treated as hydroxyl, =O, halogen, CN, COR, together with the atom to which they are attached. a , N.R. a R b , C 1~6 Haloalkyl, C 3~6 Cycloalkyl, C 6~11 Aryl, 3-7 membered heterocycloalkyl, C 1~6 Alkyl and OC 1~6 forming a 3- to 7-membered heterocycloalkyl ring optionally substituted with one or more substituents selected from alkyl; R f are independently hydrogen; as well as hydroxyl, halogen, CN, COR a , N.R. a R b , C 1~6 Haloalkyl, C 3~6 Cycloalkyl, phenyl, 3-7 membered heterocycloalkyl and OC 1~6 C optionally substituted with one or more substituents selected from alkyl 1~6 alkyl} (3) The compound of formula (1) or a deuterated form, salt, solvate or hydrate thereof, as disclosed in GB2108383.7 (Argonaut Therapeutics Limited); [ka] {In the formula, R 1A is expressed by the formula (A1), [ka] is represented by Z is =O, T, together with the carbon and nitrogen atoms therebetween (e.g., as shown in formula (A1)), is selected from monocyclic 5- to 7-membered heterocycloalkyl groups, fused bicyclic 6- to 10-membered heterocycloalkyl groups, and bridged bicyclic 6- to 9-membered heterocycloalkyl groups, wherein each of the monocyclic 5- to 7-membered heterocycloalkyl groups, fused bicyclic 6- to 10-membered heterocycloalkyl groups, and bridged bicyclic 6- to 9-membered heterocycloalkyl groups optionally contains one or more R S1 is replaced by R S1 is C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Alkoxy, C 3~12 cycloalkyl, hydroxy, halo, CN and nitro, wherein C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl and C 3~12 Cycloalkyl can each optionally be one or more R S2 is replaced by R S2 is selected from hydroxy, halo, CN and nitro. Examples include:

[0177] Any of these PRMT5 compounds can be used in the present invention.

[0178] In certain embodiments, the PRMT5 inhibitor for use in the present invention is a small molecule compound selected from the group consisting of GSK3326595 (pemurametostat), PF-6939999, JVNJ-64619178 (onametostat), LLY-283 and PRT543.

[0179] The PRMT5 inhibitor for use in the fourth or fifth aspect of the present invention can be formulated as a pharmaceutical composition.The pharmaceutical composition can include at least one pharmaceutically acceptable excipient.The dosage, route of administration and actual treatment regime can be determined by those skilled in the art.

[0180] The term "pharmaceutical acceptable excipient" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration to humans. The term "excipient" means an organic or inorganic ingredient, natural or synthetic, with which an active ingredient is combined to facilitate application. Suitable excipient types are salts, buffers, wetting agents, emulsifiers, preservatives, compatible carriers, diluents, carriers, vehicles, supplemental immunostimulants such as adjuvants, and cytokines that are well known in the art and commercially available for use in pharmaceutical preparations (see, for example, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th Ed. Mack Publishing; Kibbe et al., (2000) Handbook of Pharmaceutical Excipients, 3rd Ed., Pharmaceutical Press; and Ansel et al., (2004) Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippencott Williams and Wilkins). Optionally, the pharmaceutical composition contains one or more other therapeutic agents or compounds. Suitable pharma- ceutically acceptable excipients are relatively inert and may, for example, facilitate processing or delivery of the active compound / agent into a preparation optimized for stabilization, administration, or delivery to the body, preferably directly to the site of action.

[0181] The pharmaceutical composition may take the form of a solution, suspension, emulsion, tablet, pill, pellet, capsule, liquid-containing capsule, powder, sustained release formulation, suppository, emulsion, aerosol, spray, suspension, or any other form suitable for use.

[0182] When administered, the PRMT5 inhibitor is administered in a pharma- ceutically acceptable preparation / composition.

[0183] Administration may be enteral (e.g., oral) (i.e., the substance is given via the gastrointestinal tract) or parenteral (i.e., the substance is given by a route other than the digestive tract, such as by injection). Large biomolecules or nucleic acid molecules (e.g., certain vaccines) are typically administered parenterally by injection.

[0184] Pharmaceutical compositions for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient / drug is dissolved, suspended or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may additionally contain one or more pharma- ceutically acceptable carriers, such as antioxidants, buffers, stabilizers, preservatives, suspending agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended patient. In certain embodiments, the compositions may be lyophilized to provide a powdered form that is reconstituted as and when required. When reconstituted from a lyophilized powder, the aqueous liquid may be further diluted prior to administration. For example, diluted into an infusion bag containing 0.9% Sodium Chloride Injection, USP, or equivalent, to achieve the desired dose for administration. In certain embodiments, such administration may be performed by intravenous infusion using an intravenous (IV) device.

[0185] Preferably, the PRMT5 inhibitor is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, the active agent for IV administration is in a solution, for example, in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent. The composition for IV administration may optionally include a local anesthetic, for example, lignocaine, to ease pain at the injection site. Generally, the components are supplied separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or moisture-free concentrate, in a sealed container such as an ampule. If the PRMT5 inhibitor is administered by infusion, it may be dispensed, for example, in an infusion bottle containing pharmaceutical grade sterile water or saline. If the PRMT5 inhibitor is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the components can be mixed before administration.

[0186] The compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, prepared by conventional means with pharma- ceutically acceptable excipients, such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharma- ceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats and oils), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Preparations may also contain buffer salts, flavoring agents, coloring agents, and sweeteners as appropriate. Oral compositions can include standard vehicles, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.

[0187] The composition for use according to the present invention can be formulated in a conventional manner using one or more physiologically acceptable excipients.Therefore, the PRMT5 inhibitor and optionally another therapeutic or prophylactic agent and their physiologically acceptable salts and solvates can be formulated into pharmaceutical compositions for administration by inhalation or insufflation (either through the mouth or nose) or oral, parenteral, or mucosal (e.g., buccal, vaginal, rectal, sublingual) administration.In certain embodiments, local or systemic parenteral administration is used.

[0188] Pharmaceutical compositions for use in the therapeutic methods of the present invention are for administration in an effective amount, which is that amount of the composition that alone, or together with further doses, produces the desired response.

[0189] Suitably, the PRMT5 inhibitor may be administered as a pharmaceutical composition containing 0.1 to 1 mg, 1 to 10 mg, 10 to 50 mg, 50 to 100 mg, 100 to 500 mg, or 500 mg to 5 g of the PRMT5 inhibitor.

[0190] The preparation of suitable pharmaceutical compositions of a drug and the dosage to be administered to a subject is within the capabilities of one skilled in the art.

[0191] <Cancer> The various aspects of the present invention that use cancer cells or target methods or uses for treating cancer are applicable to any cancer.Preferably, cancer is selected from the group consisting of leukemia, lymphoma, multiple myeloma, lung cancer, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid cancer, skin cancer (including melanoma), oral squamous cell carcinoma, bladder cancer, Leydig cell tumor, biliary tract cancer, for example cholangiocarcinoma or cholangiocarcinoma, brain cancer, pancreatic cancer, colon cancer, colorectal cancer, and gynecological cancer, including ovarian cancer, endometrial cancer, fallopian tube cancer, uterine cancer and cervical cancer, including epithelial cervical cancer. In a preferred embodiment, the cancer is a leukemia and may be selected from the group consisting of acute lymphoblastic leukemia, acute myelogenous leukemia (also known as acute myeloid leukemia or acute nonlymphocytic leukemia), acute promyelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia (also known as chronic myeloid leukemia, chronic myelocytic leukemia or chronic granulocytic leukemia), chronic lymphocytic leukemia, monoblastic leukemia and hairy cell leukemia. In a further preferred embodiment, the cancer is acute lymphoblastic leukemia. In a preferred embodiment, the cancer is a lymphoma and may be selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma and small lymphocytic lymphoma.

[0192] In certain embodiments, the cancer is selected from breast cancer, esophageal cancer, bladder cancer, lung cancer, cancer of the hematopoietic system, lymphoma, medulloblastoma, rectal adenocarcinoma, colon adenocarcinoma, gastric cancer, pancreatic cancer, liver cancer, adenoid cystic carcinoma, lung adenocarcinoma, squamous cell carcinoma of the head and neck, brain tumor, hepatocellular carcinoma, renal cell carcinoma, melanoma, oligodendroglioma, ovarian clear cell carcinoma, and ovarian serous carcinoma.

[0193] In certain embodiments, the methods and uses disclosed herein provide a precision medicine approach, e.g., to target specific types of tumors, or subsets of patients with specific tumors, or specific stages of tumors, or even individual patients.

[0194] Advantageously, the method of the first or second aspect of the present invention is useful for identifying cancer patients who are most suitable for treatment with a PRMT5 inhibitor. Advantageously, such cancer treatment can achieve effective cancer treatment by preventing or treating the onset of cancer, by preventing or treating the progression of cancer, by preventing or treating the recurrence of cancer, or by preventing or treating the spread (including metastasis) of cancer.

[0195] <How it is carried out on a computer> According to a sixth aspect of the present invention there is provided a computer-implemented method of assisting in selecting a treatment for a patient with cancer, comprising the steps of: (iv) receiving a value for the level of biomarkers (a) pRb and / or (b) total E2F1 in a cancer cell-containing biological sample from the patient; (v) comparing the levels in (i) to a reference value for each biomarker; (vi) if the patient's cancer cells exhibit a decrease in the level of pRb and / or an increase in the level of total E2F1 compared to the baseline, the patient is selected for treatment with a PRMT5 inhibitor. A method is provided.

[0196] Preferably, the level is an expression level.

[0197] The term "computer-implemented" as used herein means that the method is carried out in an automated manner in a data processing unit, typically contained in a computer or similar data processing device. The data processing unit receives the value for the level of biomarker (i.e., pRb and / or (b) total E2F1). Such value may be an amount, a relative amount, or any other calculated value that reflects the amount described in detail elsewhere herein. Therefore, it should be understood that the above method does not require the determination of the amount for biomarker, but uses the value for the amount that has already been determined in advance.

[0198] The present invention also in principle contemplates a computer program, a computer program product, or a computer readable storage medium tangibly embodied with said computer program, where the computer program contains instructions which, when run on a data processing device or computer, perform the methods of the present invention as specified above. - a computer or computer network comprising at least one processor, the processor being adapted to perform a method according to one of the aspects described herein, - a computer-loadable data structure adapted to perform a method according to one of the aspects described herein while the data structure is executed on a computer, - a computer script, the computer program being adapted to perform a method according to one of the aspects described herein while the program is running on a computer, - a computer program comprising program measures for carrying out a method according to one of the aspects described herein while said computer program is running on a computer or on a computer network, a computer program comprising a program measure according to any of the above embodiments, the program measures being stored on a computer-readable storage medium, - a storage medium, on which a data structure is stored and adapted to perform a method according to one of the aspects described herein after the data structure has been loaded into a primary and / or working storage device of a computer or a computer network, - a computer program product having program code measures, which, when executed on a computer or on a computer network, can be or is stored on a storage medium for performing a method according to one of the aspects described herein, - a data stream signal, typically encrypted, containing data on the parameters defined elsewhere in this specification; - a data stream signal, typically encrypted, containing the assessment provided by the method of the present invention; The present invention further includes the following.

[0199] <Kit> The present invention also includes a kit comprising one or more tools capable of, for example, quantifying the amount of pRb and / or E2F1 biomarkers in a sample. According to a third aspect of the present invention, a kit for use in the method of the first aspect of the present invention is provided, comprising one or more reagents capable of determining the expression level of pRb and / or total E2F1. Suitably, the kit comprises an antibody or an antigen-binding portion thereof that specifically binds to E2F-1 protein, and / or an antibody or an antigen-binding portion thereof that specifically binds to pRb protein, and / or a nucleic acid oligonucleotide capable of specifically binding to RB1 transcript, and / or a nucleic acid oligonucleotide capable of specifically binding to E2F1 transcript.

[0200] In certain embodiments, the oligonucleotide capable of specifically binding to one of the biomarker transcripts is a primer or a probe. Optionally, the primer or probe is labeled, such as with a fluorescent or radioactive label.

[0201] Typically, the kit contains an antibody or antigen-binding moiety capable of binding to a biomarker in protein form (e.g., E2F1 protein or pRb protein), and / or a primer or probe capable of binding to a biomarker in nucleic acid form (e.g., E2F1 transcript or RB1 transcript). Optionally, such an antibody or antigen-binding moiety, primer or probe can be labeled, such as by fluorescence. The kit may also include instructions for use, and may contain additional elements required to carry out the method described in the instructions in the kit. The kit may also include information on data interpretation and determination, such as threshold levels for interpreting whether the level of the biomarker indicates that the patient is likely to respond well to a PRMT5 inhibitor.

[0202] The kit comprises the antibody described herein, or an epitope-binding fragment thereof. The kit may also comprise a means for obtaining a biological sample, such as a spatula or dipstick, or a container for receiving a sample. The kit may also comprise one or more assay components for detecting the amount of total E2F-1 or pRb protein. Preferably, the assay comprises an immunoassay, such as an ELISA. The kit may also comprise one or more assay components for detecting the amount of E2F-1 or RB1 transcript. Preferably, the assay is RT-PCR.

[0203] In certain embodiments, the kits also contain positive and / or negative controls. These process controls act as quality controls to ensure valid assay results.

[0204] The presence and / or amount of total E2F-1 or pRB protein in a sample can be determined by standard immunochemical techniques well known to those skilled in the art (e.g., immunohistochemistry, radioimmunoassay, ELISA, Western blot, fluorescent assay, DELFIA®, LANCE, FRET, etc.). The method can preferably be performed as a high throughput screen.

[0205] The presence and / or amount of E2F-1 or RB1 transcripts in a sample can be determined by standard nucleic acid quantification techniques well known to those of skill in the art (eg, RT-PCR, qPCR).

[0206] An increase in total E2F-1 protein or E2F-1-encoding transcripts in the patient's cancer cell sample compared to a reference value, such as the level in a normal cell sample or a normal reference value, indicates that the patient is suitable for treatment with a PRMT-5 inhibitor.

[0207] A decrease in the amount of pRb protein or RB1 transcript in the patient's cancer cell sample compared to a reference value (eg, a normal cell sample or a normal reference value) indicates that the patient is suitable for treatment with a PRMT-5 inhibitor.

[0208] Further aspects and embodiments will be apparent to those skilled in the art. All documents cited in this text are incorporated herein by reference. Aspects and embodiments of the present invention will now be described with reference to the following examples and accompanying drawings.

[0209] [array] SEQ ID NO:1 Gene name: RB1 (RB transcriptional corepressor 1) Gene number: 5925 Protein number: NP_000312.2 CCDS:CCDS31973.1 RB1 nucleotide sequence (2787nt):

[0210] SEQ ID NO:2 Translation(928aa): MPPKTPRKTAATAAAAAAEPPAPPPPPPPEEDPEQDSGPEDLPLVRLEFEETEEPDFTALCQKLKIPDHVRERAWLTWEKVSSVDGVLGGYIQKKKELWGICIFIAAVDLDEMSFT FTELQKNIEISVHKFFNLLKEIDTSTKVDNAMSRLLKKYDVLFALFSKLERTCELIYLTQPSSSISTEINSALVLKVSWITFLLAKGEVLQMEDDLVISFQLMLCVLDYFIKLSPP MLLKEPYKTAVIPINGSPRTPRRGQNRSARIAKQLENDTRIIEVLCKEHECNIDEVKNVYFKNFIPFMNSLGLVTSNGLPEVENLSKRYEEIYLKNKDLDARLFLDHDKTLQTDSI DSFETQRTPRKSNLDEEVNVIPPHTPVRTVMNTIQQLMMILNSASDQPSENLISYFNNCTVNPKESILKRVKDIGYIFKEKFAKAVGQGCVEIGSQRYKLGVRLYYRVMESMLKSE EERLSIQNFSKLLNDNIFHMSLLACALEVVMATYSRSTSQNLDSGTDLSFPWILNVLNLKAFDFYKVIESFIKAEGNLTREMIKHLERCEHRIMESLAWLSDSPLFDLIKQSKDRE GPTDHLESACPLNLPLQNNHTAADMYLSPVRSPKKKGSTTRVNSTANAETQATSFQTQKPLKSTSLSLFYKKVYRLAYLRLNTLCERLLSEHPELEHIIWTLFQHTLQNEYELMR DRHLDQIMMCSMYGICKVKNIDLKFKIIVTAYKDLPHAVQETFKRVLIKEEEYDSIIVFYNSVFMQRLKTNILQYASTRPPTLSPIPHIPRSPYKFPSSPLRIPGGNIYISPLKSP YKISEGLPTPTKMTPRSRILLVSIGESFGTSEKFQKINQMVCNSDRVLKRSAEGSNPPKPLKKLRFDIEGSDEADGSKHLPGESKFQQKLAEMTSTRTRMQKQKMNDSMDTSNKEEK

[0211] SEQ ID NO:3 Gene name: E2F1 (E2F transcription factor 1) Gene number: 1869 Protein number: NP_005216-1 CCDS:CCDS13224.1 E2F1 nucleotide sequence (1314 nt):

[0212] SEQ ID NO:4 Translation(437aa): MALAGAPAGGPCAPALEALLGAGALRLLDSSQIVIISAAQDASAPPAPTGPAAPAAGPCDPDLLLFATPQAPRPTPSAPRPALGRPPVKRRLDLETDHQYLAESSGPAR GRGRHPGKGVKSPGEKSRYETSLNLTTKRFLELLSHSADGVVDLNWAAEVLKVQKRRIYDITNVLEGIQLIAKKSKNHIQWLGSHTTVGVGGRLEGLTQDLRQLQESEQ QLDHLMNICTTQLRLLSEDTDSQRLAYVTCQDLRSIADPAEQMVMVIKAPPETQLQAVDSSENFQISLKSKQGPIDVFLCPEETVGGISPGKTPSQEVTSEEENRATDSATIVSPPPSSPPSSLTTDPSQSLLSLEQEPLLSRMGSLRAPVDEDRLSPLVAADSLLEHVREDFSGLLPEEFISLSPPHEALDYHFGLEEGEGIRDLFDCDFGDLTPLDF

[0213] SEQ ID NO:5 PRMT5 siRNA sequence CCG CUA UUG CAC CUU GGA A

[0214] SEQ ID NO:6 E2F-1 siRNA sequence: AAC UCC UCG CAG AUC GUC AUC EXAMPLES

[0215] [Example 1] The effect of PRMT5 and E2F-1 in growth control was evaluated in U2OS cells. Analysis of cell proliferation was performed, as measured by colony formation assay, and the inhibitory effect of PRMT5 was evident, with PRMT5 siRNA causing a dramatic reduction in proliferation after 10 days, which was rescued upon concomitant depletion of E2F-1 (Fig. 1a). Protein levels of PRMT5 and E2F1 are shown in Fig. 1b. Silencing PRMT5 activity by gene-specific siRNA leads to apoptotic cell death. Moreover, this cell death requires E2F1 activity, as simultaneous silencing of E2F1 and PRMT5 rescues cell death and allows cells to continue to grow (Fig. 1a).

[0216] [Example 2] Tumor cells lacking the E2F1 gene are less sensitive to AT101-mediated PRMT5 inhibition. In colorectal and breast cancer cell lines in which E2F1 has been deleted using CRISPR technology, depletion of E2F1 desensitizes the cells to PRMT5 inhibition (Figures 2(a) and (b)), indicating that E2F1 is a positive lever for the response to PRMT5 inhibition.

[0217] [Example 3] Tumor cells lacking the tumor suppressor protein pRb gene are more sensitive to PRMT5 inhibition mediated by AT101. In breast and bone cancer cell lines in which pRb has been deleted using CRISPR technology, depletion of pRb sensitizes the cells to PRMT5 inhibition (FIGS. 3(a) and (b)), indicating that pRb is a negative factor in the response to PRMT5 inhibition.

[0218] [Example 4] Tumor cells lacking the E2F1 gene are less sensitive to PRMT5 inhibition mediated by GSK3326595. In colorectal and breast cancer cell lines in which E2F1 has been deleted using CRISPR technology, depletion of E2F1 desensitized the cells to PRMT5 inhibition by GSK3326595 (FIGS. 4(a) and (b)), indicating that E2F1 is a positive lever for the response to PRMT5 inhibition mediated by GSK3326595.

[0219] [Example 5] Tumor cells lacking the E2F1 gene are less sensitive to PRMT5 inhibition mediated by PF06939999. In colorectal and breast cancer cell lines in which E2F1 has been deleted using CRISPR technology, depletion of E2F1 desensitized the cells to PRMT5 inhibition by PF06939999 (Figures 5(a) and (b)), indicating that E2F1 is a positive factor in the response to PRMT5 inhibition mediated by PF06939999.

[0220] [Example 6] Tumor cells lacking the E2F1 gene are less sensitive to PRMT5 inhibition mediated by JNJ64619178. In breast cancer cell line T-47D, in which E2F1 has been deleted using CRISPR technology, depletion of E2F1 desensitizes cells to PRMT5 inhibition by JNJ64619178 (Figure 6), indicating that E2F1 is a positive factor in the response to PRMT5 inhibition mediated by JNJ64619178.

[0221] [Example 7] Tumor cells lacking the E2F1 gene are less sensitive to LLY-283-mediated PRMT5 inhibition. In breast cancer cell line T-47D, in which E2F1 has been deleted using CRISPR technology, depletion of E2F1 desensitizes cells to LLY-283-mediated PRMT5 inhibition (Figure 7), indicating that E2F1 is a positive factor in the response to LLY-283-mediated PRMT5 inhibition.

[0222] [Example 8] Tumor cells lacking the tumor suppressor protein pRb gene are more sensitive to PRMT5 inhibition mediated by GSK3326595. In breast cancer cell lines in which pRb has been deleted using CRISPR technology, depletion of pRb sensitizes cells to PRMT5 inhibition by GSK3326595 ( FIG. 8 ), indicating that pRb is a negative factor in the response to PRMT5 inhibition mediated by GSK3326595.

[0223] [Example 9] Tumor cells lacking the tumor suppressor protein pRb gene are more sensitive to PRMT5 inhibition mediated by PF06939999. In breast cancer cell lines in which pRb has been deleted using CRISPR technology, depletion of pRb sensitizes cells to PRMT5 inhibition by PF06939999 (FIG. 9), indicating that pRb is a negative factor in the response to PRMT5 inhibition mediated by PF06939999.

[0224] [material and method] <MTTアッセイ> T-47D cells were maintained in growth medium (RPMI 1640 supplemented with 10% v / v heat-inactivated fetal bovine serum) and cultured at 37°C, 5% CO2. U2OS, MCF7 and HCT116 cells were maintained in growth medium (DMEM supplemented with 10% v / v heat-inactivated fetal bovine serum) and cultured at 37°C, 5% CO2. Under assay conditions, cells were incubated in assay medium (RPMI 1640 or DMSO supplemented with 10% v / v heat-inactivated fetal bovine serum and 100 units / mL penicillin-streptomycin) at 37°C, 5% CO2. For evaluation of the effect of compounds on the proliferation of cancer cell lines, exponentially growing cells were seeded in 96-well plates overnight at a density of 1,000 cells / well in a final volume of 100 μl of cell growth medium. The next day, cells were dosed with compounds (T-992, GSK3326595, LLY-283, PF-06939999 and JNJ64619178) (four sets of 10-point 5-fold serial dilutions in DMSO) starting at 100 μM. After compound addition, the assay plate was incubated at 37° C., 5% CO2, 90% relative humidity for 8 days. NAD(P)H-dependent cellular oxidoreductase activity was measured by adding 100 μl of thiazolyl blue tetrazolium bromide (MTT; Sigma-Aldrich) to the wells at a final concentration of 5 μM and incubated at 37° C. for 2 hours. The medium was then discarded from the wells and the formazan crystals were dissolved in DMSO (100 μl) by shaking for 15 minutes. Absorbance was read at a wavelength of 584 nM on an Omega FLUOstar plate reader (BMG Labtech Ltd, Ortenberg, Germany). Data were analyzed and ICs were calculated using MARs data analysis software (BMG Labtech Ltd, Ortenberg, Germany). 50 Values ​​were determined. The concentration of compound that inhibited cell viability by 50% was determined using a four-parameter fit of the normalized dose-response curve.

[0225] <Western Blot> Cells were harvested, washed in PBS, and resuspended in lysis buffer [50 mM Tris pH 7.4, 5 mM EDTA, 0.5% Igepal CA-630 (Sigma, Gillingham, UK), 50 mM NaF, 1 mM DTT, 0.2 mM Na3VO4, 120 mM NaCl, protease inhibitor cocktail]. Total protein concentration was determined by Bradford Assay (Bio-Rad). Whole cell lysates were prepared and lysates were separated by 4-20% SDS-PAGE (Bio-Rad) and transferred to PVDF membranes (GE Healthcare, Piscataway, NJ). Membranes were blocked with 5% milk in PBS containing 0.2% Tween 20 for 1 h at room temperature. Primary antibodies for actin (Sigma, A2228), E2F1 (CST, #3742) and Rb (#9309) were diluted 1:1000 in 5% milk and incubated overnight at 4° C. Membranes were washed with PBS-T and incubated with HRP-conjugated secondary antibodies (CST) diluted in PBS-T with 5% milk for 1 h at room temperature, washed and developed using SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific).

[0226] <Colony formation assay> U2OS cells were seeded in 6-well plates at a density of 1000 cells / well, and siRNA transfection was performed as described in the following section. U2OS cells were allowed to establish colonies over a period of 10 days, after which the experiment was terminated. To avoid physical damage to the cells, the culture medium was gently aspirated, and the plates were briefly rinsed with PBS. Crystal violet (Sigma-Aldrich) dye (0.5%) was applied to the cells for 2 minutes, followed by rinsing with autoclaved deionized water and air drying. The plates were scanned, colonies were measured, and counted using a Gelcount™ Colony Counter (Oxford Optronics).

[0227] <siRNA Transfection> Oligofectamine reagent (Invitrogen) and siRNA complexes (non-targeting, E2F1, and PRMT5) were incubated separately with OPTI-MEM® I Reduced Serum Media (Gibco®) at room temperature for 5 minutes. The two mixtures were combined and incubated for an additional 20 minutes. The siRNA transfection mixture was added dropwise to U2OS cells. A commercially available non-targeting siRNA control was obtained from Dharmacon. PRMT5 siRNA sequence: 5’-CCG CUA UUG CAC CUU GGA A-3’ (SEQ ID NO: 5), E2F-1 siRNA sequence: 5’-AAC UCC UCG CAG AUC GUC AUC-3’ [sense strand shown] (SEQ ID NO: 6). In experiments involving treatment with two or more siRNAs, non-targeting siRNA was included to ensure that equal amounts of siRNA were transfected across all samples.

[0228] <Gene Disruption by CRISPR-Cas9> For the generation of E2F1 and pRb knockout cell lines using CRISPR-Cas9 technology, pSpCas9(BB)-2A-Puro (Addgene plasmid #48139) was used. Cell lines were generated as described in (Ran et al., 2013).

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Claims

1. 1. A method of selecting a treatment for a patient with cancer, comprising: (i) determining the expression level of biomarkers (a) pRb and / or (b) total E2F1 in a cancer cell-containing biological sample isolated from said patient; (ii) comparing the expression levels in (i) with a reference value for each biomarker; wherein the patient is selected for treatment with a PRMT5 inhibitor if the patient's cancer cells exhibit decreased expression of pRb and / or increased expression of total E2F1 compared to the reference value. method.

2. The method of claim 1 , wherein the expression level is protein or nucleic acid expression.

3. The method of claim 1 , wherein the determination is based on the expression level of total E2F1 protein.

4. The method of claim 1 , wherein the determination is based on the expression level of pRb protein.

5. The method of claim 1 , wherein the determination is based on expression levels of pRb and total E2F1.

6. The method of claim 1 , wherein the level of a protein is detected using an antibody that binds to the protein.

7. The method of claim 6 , wherein the antibody is a monoclonal antibody.

8. The method of claim 1, wherein the reduced expression level of pRb is determined indirectly by identifying an RB1 mutation indicative of reduced or absent expression in the cancer cells.

9. 1. A method for selecting a treatment for a patient having cancer, comprising determining whether the PRB1 gene in cancer cells contains one or more mutations that result in reduced expression, including no expression, of pRb, and if the cancer cells contain one or more mutations in the PRB1 gene that result in reduced expression of pRb, the patient is selected for treatment with a PRMT5 inhibitor.

10. 10. The method of claim 1 or 9, wherein the cancer is selected from the group consisting of leukemia; lymphoma; multiple myeloma; lung cancer; liver cancer; breast cancer; head and neck cancer; neuroblastoma; thyroid cancer; skin cancer (including melanoma); oral squamous cell carcinoma; bladder cancer; Leydig cell tumor; biliary tract cancer, e.g., cholangiocarcinoma or bile duct carcinoma; brain cancer; pancreatic cancer; colon cancer; colorectal cancer; and gynecological cancers, including ovarian cancer, endometrial cancer, fallopian tube cancer, uterine cancer, and cervical cancer, including epithelial cervical cancer.

11. A kit for identifying cancers that may be susceptible to treatment by inhibition of PRMT5, comprising an antibody or antigen-binding portion thereof that specifically binds to E2F-1 protein, and / or an antibody or antigen-binding portion thereof that specifically binds to pRb protein, and / or a nucleic acid oligonucleotide capable of specifically binding to an RB1 transcript, and / or a nucleic acid oligonucleotide capable of specifically binding to an E2F1 transcript.

12. 12. The kit of claim 11, further comprising assay reagents for detection of antibody target binding or nucleic acid oligonucleotide target binding, and / or instructions for use.

13. A pharmaceutical composition for treating cancer in which cancer cells express higher than normal levels of total E2F1 protein and / or reduced levels of pRb protein compared to normal, comprising a PRMT5 inhibitor.

14. 14. The pharmaceutical composition of claim 13, wherein the cancer cells express higher than normal levels of total E2F1 protein.

15. The pharmaceutical composition of claim 13 , wherein the cancer cells express reduced levels of pRb protein compared to normal cells.

16. 14. The pharmaceutical composition of claim 13, wherein the cancer cells express higher than normal levels of total E2F1 protein and reduced levels of pRb protein compared to normal.

17. A pharmaceutical composition for treating a pRb-deficient cancer or for treating a cancer in which the cancer cells express reduced levels of pRb protein compared to normal, comprising a PRMT5 inhibitor.

18. The pharmaceutical composition described in claim 13 or 17, wherein the PRMT5 inhibitor is selected from the group consisting of an antibody, an RNA interference molecule, an antisense oligonucleotide or a small molecule compound.

19. The pharmaceutical composition of claim 18, wherein the small molecule PRMT5 inhibitor is selected from GSK3326595 (pemurametostat), PF-6939999, JNJ-64619178 (onametostat), and LLY-283.

20. 18. The pharmaceutical composition of claim 13 or 17, wherein the cancer is selected from the group consisting of leukemia; lymphoma; multiple myeloma; lung cancer; liver cancer; breast cancer; head and neck cancer; neuroblastoma; thyroid cancer; skin cancer (including melanoma); oral squamous cell carcinoma; bladder cancer; Leydig cell tumor; biliary tract cancer, e.g., cholangiocarcinoma or bile duct carcinoma; brain cancer; pancreatic cancer; colon cancer; colorectal cancer; and gynecological cancers, including ovarian cancer, endometrial cancer, fallopian tube cancer, uterine cancer and cervical cancer, including epithelial cervical cancer.

21. 18. The pharmaceutical composition of claim 17, wherein the cancer is selected from breast cancer, esophageal cancer, bladder cancer, lung cancer, cancer of the hematopoietic system, lymphoma, medulloblastoma, rectal adenocarcinoma, colon adenocarcinoma, gastric cancer, pancreatic cancer, liver cancer, adenoid cystic carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, brain tumor, hepatocellular carcinoma, renal cell carcinoma, melanoma, oligodendroglioma, ovarian clear cell carcinoma, and ovarian serous carcinoma.

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