Treatment of prostate cancer by promoting PDE4D7

Abstract

The present invention relates to a product for use in the treatment, prevention, or remission of prostate cancer in subjects requiring treatment, prevention, or remission, wherein the product induces the expression of PDE4D7 or promotes the activity of phosphodiesterase 4D7. The product may be a direct or indirect activator of PDE4D7 activity, or a product for causing or promoting the expression of PDE4D7 in a subject. The present invention further specifies kits of elements and the in vitro or ex vivo use of the product.

Description

[Technical Field]

[0001] The present invention relates to a product for use in the treatment, prevention, or remission of prostate cancer by reducing therapy resistance in subjects requiring treatment, prevention, or remission. In particular, the product is used to induce the expression of PDE4D7 or to enhance the activity of phosphodiesterase 4D7. [Background technology]

[0002] Cancer is a class of diseases characterized by uncontrolled growth, invasion, and, in some cases, metastasis of a group of cells. These three malignant characteristics of cancer differentiate it from benign tumors, which are self-limiting and do not invade or metastasize. In men, the three most commonly diagnosed cancers in developed countries are prostate, lung, and colorectal cancer. Prostate cancer, in particular, is the most common malignant tumor in European men. In 2002, an estimated 225,000 men in Europe were newly diagnosed with prostate cancer, and approximately 83,000 died from the disease.

[0003] For example, prostate cancer is traditionally diagnosed by serum levels of prostate-specific antigen (PSA). However, PSA is not specific to prostate cancer and can be elevated in other environments, leading to numerous false positives (approximately 70% of men who undergo biopsy and have elevated PSA levels do not have cancer). Furthermore, there is an unpredictable number of false negatives in the presence of "normal" PSA tests, resulting in the later development of prostate cancer.

[0004] In patients receiving treatment, the most important clinical prognostic indicators of disease outcome are grade, pre-therapy PSA level, and Gleason score. Higher grades and stages are associated with a poorer prognosis. Nomograms can be used to calculate the estimated risk for individual patients. Predictions are based on the experience of a large number of patients.

[0250] A complicating factor is that the majority of patients have multiple independent tumor foci at diagnosis, and these foci have independent genetic alterations and molecular characteristics. This widespread heterogeneity between foci poses a risk that prognosis will be based on the wrong tumor foci.

[0005] Androgen deprivation therapy induces remission in 80–90% of patients treated, resulting in a median progression-free survival of 12–33 months. Following remission, an androgen-independent phenotype typically develops, with a median overall survival of 23–37 months from the start of androgen deprivation therapy. It is unclear how androgen independence is established and how it re-establishes progression. Androgen deprivation therapy includes, for example, the administration of anti-androgen drugs.

[0006] US2012 / 129788A1 relates to the 4D7 isoform of PDE and teaches that tumors have increased PDE4D7 expression, which can be used for diagnostic purposes.

[0007] EP3434787A1 teaches that PDE4D7 expression is reduced in hormone resistance and that PDE4D7 expression levels should be used for diagnosis.

[0008] EP3303618A1 relates to a method for determining a gene expression signature for diagnosing a patient with prostate cancer, for example, for establishing a prognosis for such a patient, for example for determining the predisposition of such a patient to develop high-grade or low-grade disease after first-line treatment, and / or for identifying and stratifying patients for available therapies.

[0009] Henderson et al. (BRITISH JOURNAL OF CANCER, Vol. 110, No. 5, February 11, 2014 (2014-02-11), pp. 1278-1287) describe the downregulation of PDE4D7 in androgen-independent prostate cancer cells.

[0010] Boettcher et al. (ONCOTARGET, Vol. 7, No. 43, October 25, 2016 (2016-10-25), pp. 70669-70684) describe how the PDE4D isoform composition is deregulated in primary prostate cancer and serves as an indicator of disease progression and the development of distant metastasis. [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] Therefore, there is always a need for novel and improved methods for treating cancers such as prostate cancer. Furthermore, there is always a need for methods to reduce or prevent resistance to treatments such as anti-androgen drugs or PARP inhibitors.

[0012] These drawbacks are overcome, in particular, by the products and methods outlined in the appended claims. [Means for solving the problem]

[0013] In a first aspect, the present invention relates to a product for use in the treatment, prevention, or remission of prostate cancer by reducing therapy resistance in subjects requiring treatment, prevention, or remission, wherein the product induces the expression of PDE4D7 or promotes the activity of phosphodiesterase 4D7, and is selected from the following: A polynucleotide encoding a peptide having the sequence of SEQ ID NO: 2, or a variant thereof, wherein the variant is a polynucleotide encoding a peptide having a sequence having at least 90% sequence homology to SEQ ID NO: 2, and the peptide has phosphodiesterase activity, or A polynucleotide comprising the nucleotide sequence defined by SEQ ID NO: 1, or a variant thereof, wherein the variant is a polynucleotide comprising a nucleotide sequence having at least 90% sequence homology to SEQ ID NO: 1, and the polynucleotide encodes a peptide having phosphodiesterase activity, a variant, or A peptide comprising the peptide sequence defined by SEQ ID NO: 2, or a variant thereof, wherein the variant is a peptide comprising a peptide sequence having at least 90% sequence homology to SEQ ID NO: 2, and the peptide has phosphodiesterase activity, a variant, or A peptide comprising the peptide sequence encoded by a polynucleotide comprising the nucleotide sequence defined by SEQ ID NO: 1, or a variant thereof, wherein the variant is a peptide comprising a peptide sequence encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence homology to SEQ ID NO: 1, and the polynucleotide encodes a peptide having phosphodiesterase activity, a variant, or A compound defined by formula I, wherein

Chemical formula

[0014] In a second aspect, the present invention relates to a compound for use in the treatment, prevention, or remission of prostate cancer in subjects requiring treatment, prevention, or remission, wherein the compound is an ATR inhibitor or a metabolic inhibitor, and the use is When high expression of PDE4D7 is observed in tumors, Low expression of PDE4D7 is observed, but PDE4D7 activity does not increase sufficiently to make the tumor sensitive again to inhibitor treatment, or In the case of metastatic tumors The present invention relates to a compound for use, comprising administering the aforementioned compound to a person. [Brief explanation of the drawing]

[0015] [Figure 1A] This figure shows the differences in production and PCa-related genes in PDE4D7 knockdown LNCaP cell lines. A) PDE4D7 mRNA expression by qRT-PCR. Doubling of LNCaP WT (2-ΔΔCt) for shRNA-scrambled (SC2) and shRNA-PDE4D7 knockdown (P1). Error bars represent mean + / - SEM, N=3, *p<0.05, ****p<0.0001. [Figure 1B]This figure shows the differences in production and PCa-related genes in PDE4D7 knockdown LNCaP cell lines. B) PDE4D7 protein expression in WT and shRNA-PDE4D7 knockdown (P1) LNCaP. Error bars represent mean + / - SEM, N=3, *p<0.05, ****p<0.0001. [Figure 1C] This figure shows the differences in production and PCa-related genes in PDE4D7 knockdown LNCaP cell lines. C) Immunocytochemical staining of PDE4D7 protein expression in WT and P1 LNCaP cells (yellow). [Figure 1D] This figure shows the differences in production and PCa-related genes in PDE4D7 knockdown LNCaP cell lines. D) Heatmap of z-scores for top genes specifically expressed between WT and P1 LNCaP cells, as determined by RNA sequencing. [Figure 1E] This figure shows the differences in production and PCa-related genes in PDE4D7 knockdown LNCaP cell lines. E) Log-fold changes (FC) of higher-level genes altered in the PCa-related pathway. [Figure 1F] This figure shows the differences in production and PCa-related gene expression in PDE4D7 knockdown LNCaP cell lines. F) Western blot and quantification of protein expression of PCa-related genes identified by RNA-seq in WT and P1 LNCaP cells. Error bars represent mean + / - SEM, N=3, *p<0.05, ****p<0.0001. [Figure 2A] This figure shows that PDE4D7 knockdown in LNCaP cells enhances proliferation and confers resistance to enzalutamide. A) Real-time proliferation of LNCaP WT, shRNA-scrambled (SC2), and shRNA-PDE4D7 knockdown (P1) cells. Gradient analysis by linear regression. Error bars represent mean + / - SEM, N=3, **p<0.01, ****p<0.0001. [Figure 2B]This figure shows that PDE4D7 knockdown in LNCaP cells enhances proliferation and confers resistance to enzalutamide. Real-time analysis of LNCaP WT (B) and shRNA-PDE4D7 knockdown (P1) cells (C) with B&C enzalutamide (0 μM to 30 μM) or 0.1% DMSO. Error bars represent mean + / - SEM, N=3, **p<0.01, ****p<0.0001. [Figure 2C] This figure shows that PDE4D7 knockdown in LNCaP cells enhances proliferation and confers resistance to enzalutamide. Real-time analysis of LNCaP WT (B) and shRNA-PDE4D7 knockdown (P1) cells (C) with B&C enzalutamide (0 μM to 30 μM) or 0.1% DMSO. Error bars represent mean + / - SEM, N=3, **p<0.01, ****p<0.0001). [Figure 2D] This figure shows that PDE4D7 knockdown in LNCaP cells enhances proliferation and confers resistance to enzalutamide. D) Western blot and quantification of AR-V7 protein expression in WT and P1 LNCaP cells. Error bars represent mean + / - SEM, N=3, **p<0.01, ****p<0.0001). [Figure 2E] This figure shows that PDE4D7 knockdown in LNCaP cells enhances proliferation and confers resistance to enzalutamide. Western blot and quantification of E) cyclin B1 and F) cyclin D1 protein expression in WT and P1 LNCaP cells. Error bars represent mean + / - SEM, N=3, **p<0.01, ****p<0.0001). [Figure 2F] This figure shows that PDE4D7 knockdown in LNCaP cells enhances proliferation and confers resistance to enzalutamide. Western blot and quantification of E) cyclin B1 and F) cyclin D1 protein expression in WT and P1 LNCaP cells. Error bars represent mean + / - SEM, N=3, **p<0.01, ****p<0.0001). [Figure 3A]This figure shows that PDE4D7 knockdown in LNCaP cells enhances resistance to olaparib. A&B) Real-time proliferation of LNCaP WT (A) and shRNA-PDE4D7 knockdown (P1) cells after treatment with olaparib (0 μM to 30 μM) or 0.1% DMSO. Cell indices normalized to treatment time. Gradient analysis by linear regression over 0 to 30 h. Error bars represent mean ± SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 3B] This figure shows that PDE4D7 knockdown in LNCaP cells enhances resistance to olaparib. A&B) Real-time proliferation of LNCaP WT (A) and shRNA-PDE4D7 knockdown (P1) cells after treatment with olaparib (0 μM to 30 μM) or 0.1% DMSO. Cell indices normalized to treatment time. Gradient analysis by linear regression over 0 to 30 h. Error bars represent mean ± SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 3C] This figure shows that PDE4D7 knockdown in LNCaP cells enhances resistance to olaparib. C) Western blot and quantification of PARP and cleaved PARP (cPARP) protein expression between WT and P1 LNCaP cells. Error bars represent mean ± SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 3D] This figure shows that PDE4D7 knockdown in LNCaP cells enhances resistance to olaparib. D) Real-time proliferation of P1 and LNCaP P1-PDE4D7+ / + transient reexpression cells in response to 0.1% DMSO (D), 10 μM olaparib (E), or 10 μM docetaxel (F). Cell indices normalized to treatment time. Gradient analysis from 0 to 48 h post-treatment. Error bars represent mean ± SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 3E]This figure shows that PDE4D7 knockdown in LNCaP cells enhances resistance to olaparib. D) Real-time proliferation of P1 and LNCaP P1-PDE4D7+ / + transient reexpression cells in response to 0.1% DMSO (D), 10 μM olaparib (E), or 10 μM docetaxel (F). Cell indices normalized to treatment time. Gradient analysis from 0 to 48 h post-treatment. Error bars represent mean ± SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 3F] This figure shows that PDE4D7 knockdown in LNCaP cells enhances resistance to olaparib. D) Real-time proliferation of P1 and LNCaP P1-PDE4D7+ / + transient reexpression cells in response to 0.1% DMSO (D), 10 μM olaparib (E), or 10 μM docetaxel (F). Cell indices normalized to treatment time. Gradient analysis from 0 to 48 h post-treatment. Error bars represent mean ± SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4A] This figure shows that DNA damage-inducing and repair agents affect both WT and PDE4D7 knockdown LNCaP (P1). Real-time proliferation analysis of docetaxel (DMSO: control (0.1%); DOC_5: 5 μM docetaxel; DOC_7.5: 7.5 μM docetaxel; DOC_10: 10 μM docetaxel; DOC_25: 25 μM docetaxel; treatment of WT (A) or P1 (B) LNCaP, or P1 and (C) P1-PDE4D7+ / + (after transient PDE4D7 reexpression) LNCaP) including gradient analysis from 0 to 48 hours after treatment. Error bars represent mean + / - SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4B]This figure shows that DNA damage-inducing and repair agents affect both WT and PDE4D7 knockdown LNCaP (P1). Real-time proliferation analysis of docetaxel (DMSO: control (0.1%); DOC_5: 5 μM docetaxel; DOC_7.5: 7.5 μM docetaxel; DOC_10: 10 μM docetaxel; DOC_25: 25 μM docetaxel; treatment of WT (A) or P1 (B) LNCaP, or P1 and (C) P1-PDE4D7+ / + (after transient PDE4D7 reexpression) LNCaP) including gradient analysis from 0 to 48 hours after treatment. Error bars represent mean + / - SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4C] This figure shows that DNA damage-inducing and repair agents affect both WT and PDE4D7 knockdown LNCaP (P1). Real-time proliferation analysis of docetaxel (DMSO: control (0.1%); DOC_5: 5 μM docetaxel; DOC_7.5: 7.5 μM docetaxel; DOC_10: 10 μM docetaxel; DOC_25: 25 μM docetaxel; treatment of WT (A) or P1 (B) LNCaP, or P1 and (C) P1-PDE4D7+ / + (after transient PDE4D7 reexpression) LNCaP) including gradient analysis from 0 to 48 hours after treatment. Error bars represent mean + / - SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4D] This figure shows that DNA damage-inducing and repair agents affect both WT and PDE4D7 knockdown LNCaP(P1) cells. Real-time proliferation of LNCaP WT(D) and P1(E) cells by seracertib (0 μM to 30 μM). Gradient analysis from 0 to 30 h after treatment. Cell indices normalized to treatment time. Error bars represent mean + / - SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4E]This figure shows that DNA damage-inducing and repair agents affect both WT and PDE4D7 knockdown LNCaP(P1) cells. Real-time proliferation of LNCaP WT(D) and P1(E) cells by seracertib (0 μM to 30 μM). Gradient analysis from 0 to 30 h after treatment. Cell indices normalized to treatment time. Error bars represent mean + / - SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 5A] This figure shows that PDE4D7 knockdown alters the LNCaP response to various compounds. A) Western blot and quantification of glucocorticoid receptor (GR) protein expression between WT and PDE4D7 knockdown P1 LNCaP. Error bars represent mean + / - SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 5B] This figure shows that PDE4D7 knockdown alters the LNCaP response to various compounds. Real-time growth of WT(B) and P1(C)LNCaP during treatment with 0–30 μM mifepristone or 0.1% DMSO (control), including gradient analysis from 0–48 hours after treatment with B and C). Error bars represent mean + / - SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 5C] This figure shows that PDE4D7 knockdown alters the LNCaP response to various compounds. Real-time growth of WT(B) and P1(C)LNCaP during treatment with 0–30 μM mifepristone or 0.1% DMSO (control), including gradient analysis from 0–48 hours after treatment with B and C). Error bars represent mean + / - SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 5D]This figure shows that PDE4D7 knockdown alters the LNCaP response to various compounds. Real-time growth of WT(D) and PDE4D7 knockdown P1(E)LNCaP during treatment with 1-10 μM mebendazole or 0.1% DMSO, including gradient analysis from 0-48 hours after D&E treatment. Error bars represent mean + / - SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 5E] This figure shows that PDE4D7 knockdown alters the LNCaP response to various compounds. Real-time growth of WT(D) and PDE4D7 knockdown P1(E)LNCaP during treatment with 1-10 μM mebendazole or 0.1% DMSO, including gradient analysis from 0-48 hours after D&E treatment. Error bars represent mean + / - SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 6A] This figure shows the real-time proliferation analysis of WT (A) and PDE4D7 knockdown P1 (B) cells during treatment with metformin (0-25 μM), including gradient analysis from 0 to 48 hours after treatment (A & B). Error bars represent mean + / - SEM, N=3, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 6B] This figure shows the real-time proliferation analysis of WT (A) and PDE4D7 knockdown P1 (B) cells during treatment with metformin (0-25 μM), including gradient analysis from 0 to 48 hours after treatment (A & B). Error bars represent mean + / - SEM, N=3, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 6C] This figure shows the real-time proliferation analysis of WT (C) and PDE4D7 knockdown P1 (D) cells after treatment with the PDE4 activator ML-R2 (1-10 μM), including gradient analysis from 0 to 48 hours after C&D treatment. Error bars represent mean + / - SEM, N=3, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 6D]This figure shows the real-time proliferation analysis of WT (C) and PDE4D7 knockdown P1 (D) cells after treatment with the PDE4 activator ML-R2 (1-10 μM), including gradient analysis from 0 to 48 hours after C&D treatment. Error bars represent mean + / - SEM, N=3, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 7A] This figure shows the differences in PDE4D isoform mRNA expression among knockdown LNCaP cell lines. RT-qPCR of various PDE4D knockdown LNCaP cell lines shows a doubling of gene expression compared to WT(2-ΔΔCt) for PDE4D5(A), PDE4D7(B), and PDE4D9(C). One-way ANOVA was performed on the ΔΔCt values. Error bars represent the mean ± SEM for N=3 (*=p≦0.05, **=p≦0.01, ***=p≦0.001). [Figure 7B] This figure shows the differences in PDE4D isoform mRNA expression among knockdown LNCaP cell lines. RT-qPCR of various PDE4D knockdown LNCaP cell lines shows a doubling of gene expression compared to WT(2-ΔΔCt) for PDE4D5(A), PDE4D7(B), and PDE4D9(C). One-way ANOVA was performed on the ΔΔCt values. Error bars represent the mean ± SEM for N=3 (*=p≦0.05, **=p≦0.01, ***=p≦0.001). [Figure 7C] This figure shows the differences in PDE4D isoform mRNA expression among knockdown LNCaP cell lines. RT-qPCR of various PDE4D knockdown LNCaP cell lines shows a doubling of gene expression compared to WT(2-ΔΔCt) for PDE4D5(A), PDE4D7(B), and PDE4D9(C). One-way ANOVA was performed on the ΔΔCt values. Error bars represent the mean ± SEM for N=3 (*=p≦0.05, **=p≦0.01, ***=p≦0.001). [Figure 8]This figure shows genomic changes identified by RNA-seq between WT and scrambled-shRNA(SC2)LNCaP. Western blot and quantification of PCa-related RNA-seq gene protein expression in WT and SC2 LNCaP (N=1). [Figure 9A] This figure shows that re-expression of PDE4D7 reduces proliferation. (A) Real-time proliferation analysis of PDE4D7 knockdown P1 and PDE4D7 knockdown cells with stable reintroduction of PDE4D7 P1-4D7+ cells. [Figure 9B] This figure shows that re-expression of PDE4D7 reduces proliferation. (B) Gradient analysis from 0 to 72 hours after the start. Error bars represent mean ± SEM for N=3, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 9C] This figure shows that re-expression of PDE4D7 reduces cell proliferation. (C) Western blot of protein expression of PDE4D7 isoforms in P1-4D7+ and P1 cells. [Figure 10A] This figure shows the results of a study on the real-time proliferation of prostate cancer cells using the PDE4 activating compound MR-L2; (i) real-time proliferation; (ii) gradient analysis of real-time proliferation. A) WT_DMSO: LNCaP wild-type DMSO control (0.1%); WT_ML-R2_1: LNCaP wild-type with 1 μM ML-R2; WT_ML-R2_10: LNCaP wild-type with 10 μM ML-R2. Error bars represent mean ± SEM for N=3, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 10B]This figure shows the results of a study on the real-time proliferation of prostate cancer cells using the PDE4 activating compound MR-L2; (i) real-time proliferation; (ii) gradient analysis of real-time proliferation. B) P1_DMSO: LNCaP PDE4D7 knockdown cell line P1 DMSO control (0.1%); P1_ML-R2_1: LNCaP PDE4D7 knockdown cell line P1 with 1 μM ML-R2; P1_ML-R2_10: LNCaP PDE4D7 knockdown cell line P1 with 10 μM ML-R2. Error bars represent mean ± SEM for N=3, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 10C] This figure shows the effects of the PDE4 activating compound MR-L2 on the real-time proliferation of prostate cancer cells; (i) real-time proliferation; (ii) gradient analysis of real-time proliferation. C) LNCaP PDE4D7 knockdown cell line P1 by transient reexpression of P14D7+ / +_DMSO:PDE4D7 DMSO control (0.1%); LNCaP PDE4D7 knockdown cell line P1 by transient reexpression of P14D7+ / +_DMSO:PDE4D7 and ML-R2 (10 μM). Error bars represent mean ± SEM for N=3, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 11A] This figure shows the results of a study on the real-time proliferation of prostate cancer cells using the PARP inhibitor olaparib. A) P1_OLA: LNCaP PDE4D7 knockdown cell line P1 with 10 μM olaparib; P1+4D7_OLA: Transient re-expression of PDE4D7 with 10 μM olaparib; LNCaP PDE4D7 knockdown cell line P1 with 10 μM olaparib; P1+4D7_DMSO: LNCaP PDE4D7 knockdown cell line P1 with transient re-expression of PDE4D7 DMSO control (0.1%). [Figure 11B] This figure shows the results of a study on the real-time proliferation of prostate cancer cells with the PARP inhibitor olaparib. B) Gradient analysis of real-time proliferation. Error bars represent mean ± SEM for N=3, with **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 12]This figure shows the ratio of PARP and cleaved PARP (PARPc) as determined by Western blot analysis in LNCaP WT and LNCaP PDE4D7 knockdown cell line P1 when incubated with 0.1% DMSO (control), 10 μM olaparib, and 10 nM docetaxel, including quantification of Western blot bands normalized to DMSO. [Figure 13A] This figure shows a survival analysis of the pPDE4D7 score against prostate cancer-specific mortality (PCSM) after the initiation of salvage radiotherapy (SRT). Two cutoffs for the pPDE4D7 score were defined by AUROC (Area Under the ROC Curve) analysis, with prostate cancer-specific mortality over 5 years after the initiation of SRT as the dependent variable and the pPDE4D7 score as the independent variable. One cutoff (pPDE4D7>0.2) was defined as the point in the AUROC with maximum sensitivity and specificity. The cutoff was defined as the point with maximum sensitivity (pPDE4D7>0.87). As a result, the pPDE4D7 score classified stratified patients into three subcohorts: pPDE4D7 score>0.87: "high"; pPDE4D7 score>0.2 and <=0.87: "medium"; and pPDE4D7 score <=0.2: "low". (A) Kaplan-Meier survival analysis of time to prostate cancer-specific death after initiation of SRT in a cohort of clinically high-risk prostate cancer patients (N=348). Patients were stratified according to their PDE4D7 score, and higher scores were associated with a lower risk of death from prostate cancer compared to lower PDE4D7 scores. Log-rank p-values ​​are shown. [Figure 13B]This figure shows a survival analysis of the pPDE4D7 score against prostate cancer-specific mortality (PCSM) after the initiation of salvage radiotherapy (SRT). Two cutoffs for the pPDE4D7 score were defined by AUROC (Area Under the ROC Curve) analysis, with prostate cancer-specific mortality over 5 years after the initiation of SRT as the dependent variable and the pPDE4D7 score as the independent variable. One cutoff (pPDE4D7>0.2) was defined as the point in the AUROC with maximum sensitivity and specificity. The cutoff was defined as the point with maximum sensitivity (pPDE4D7>0.87). As a result, the pPDE4D7 score classified stratified patients into three subcohorts: pPDE4D7 score>0.87: "high"; pPDE4D7 score>0.2 and <=0.87: "medium"; and pPDE4D7 score <=0.2: "low". (B) Kaplan-Meier survival analysis of time to all-cause death after initiation of SRT in a cohort of clinically high-risk prostate cancer patients (N=348). Log-rank p-values ​​are shown. [Figure 13C] This figure shows a survival analysis of the pPDE4D7 score against prostate cancer-specific mortality (PCSM) after the initiation of salvage radiotherapy (SRT). Two cutoffs for the pPDE4D7 score were defined by AUROC (Area Under the ROC Curve) analysis, with prostate cancer-specific mortality over 5 years after the initiation of SRT as the dependent variable and the pPDE4D7 score as the independent variable. One cutoff (pPDE4D7>0.2) was defined as the point in the AUROC with maximum sensitivity and specificity. The cutoff was defined as the point with maximum sensitivity (pPDE4D7>0.87). As a result, the pPDE4D7 score classified stratified patients into three subcohorts: pPDE4D7 score>0.87: "high"; pPDE4D7 score>0.2 and <=0.87: "medium"; and pPDE4D7 score <=0.2: "low". (C) Kaplan-Meier survival analysis of time to prostate cancer-specific death after initiation of SRT in a clinically high-risk prostate cancer patient cohort (N=348). The patient group was stratified according to the EAU-BCR risk score, which stratifies patients into low vs. high-risk classes. Log-rank p-values ​​are shown. [Figure 13D] This figure shows a survival analysis of the pPDE4D7 score against prostate cancer-specific mortality (PCSM) after the initiation of salvage radiotherapy (SRT). Two cutoffs for the pPDE4D7 score were defined by AUROC (Area Under the ROC Curve) analysis, with prostate cancer-specific mortality over 5 years after the initiation of SRT as the dependent variable and the pPDE4D7 score as the independent variable. One cutoff (pPDE4D7>0.2) was defined as the point in the AUROC with maximum sensitivity and specificity. The cutoff was defined as the point with maximum sensitivity (pPDE4D7>0.87). As a result, the pPDE4D7 score classified stratified patients into three subcohorts: pPDE4D7 score>0.87: "high"; pPDE4D7 score>0.2 and <=0.87: "medium"; and pPDE4D7 score <=0.2: "low". (D) Kaplan-Meier survival analysis after SRT for time to prostate cancer-specific death after initiation of ADT in a cohort of N=179 prostate cancer patients. Log-rank p-values ​​are shown. [Figure 13E] This figure shows a survival analysis of the pPDE4D7 score against prostate cancer-specific mortality (PCSM) after the initiation of salvage radiotherapy (SRT). Two cutoffs for the pPDE4D7 score were defined by AUROC (Area Under the ROC Curve) analysis, with prostate cancer-specific mortality over 5 years after the initiation of SRT as the dependent variable and the pPDE4D7 score as the independent variable. One cutoff (pPDE4D7>0.2) was defined as the point in the AUROC with maximum sensitivity and specificity. The cutoff was defined as the point with maximum sensitivity (pPDE4D7>0.87). As a result, the pPDE4D7 score classified stratified patients into three subcohorts: pPDE4D7 score>0.87: "high"; pPDE4D7 score>0.2 and <=0.87: "medium"; and pPDE4D7 score <=0.2: "low". (E) Kaplan-Meier survival analysis after SRT for time to all-cause mortality after the start of ADT in a cohort of N=179 prostate cancer patients. Log-rank p-values ​​are shown. [Figure 14]This figure shows Western blotting and quantification of AR-V7 expression in WT and P1 LNCaP. Mean + / - SEM, N=3, *p<0.05, ****p<0.0001. [Figure 15A] This figure shows that PDE4D7 knockdown enhances proliferation and confers enzalutamide resistance, which is then rescued upon PDE4D7 reexpression. (A) Real-time proliferation analysis of PDE4D7 in response to P1-TET4D7+-doxycycline induction alone, or (B) in response to 10 μM enzalutamide. [Figure 15B] This figure shows that PDE4D7 knockdown enhances proliferation and confers enzalutamide resistance, which is then rescued upon PDE4D7 reexpression. (A) Real-time proliferation analysis of PDE4D7 in response to P1-TET4D7+-doxycycline induction alone, or (B) in response to 10 μM enzalutamide. [Figure 16] This is a box plot of BRCA2 gene expression in human clinical patient samples, categorized by PDE4D7 expression classes. Gene expression is obtained after TPM calculation based on RNA-seq counting data. The PDE4D7 classes represent various categories of PDE4D7 expression based on PDE4D7 scores (see references), with PDE4D7_Class 1 (leftmost box) representing the lowest PDE4D7 score (i.e., lowest PDE4D7 expression), and PDE4D7_Class 4 (rightmost box) representing the highest PDE4D7 score (i.e., highest PDE4D7 expression). The number of patients per class is: PDE4D7_Class 1 (N=13); PDE4D7_Class 2 (N=134); PDE4D7_Class 3 (N=301); PDE4D7_Class 4 (N=85). The median expression for each group is indicated by the bar within each box. Red crosses represent the mean expression levels for each group. Circles represent outlier expression levels. The p-values ​​were calculated using the ANOVA Kruskal-Wallis test and represent significant changes in expression across the four PDE4D7 classes. [Figure 17A](A&B) This figure shows the real-time proliferation of LNCaP WT(E) and P1(F) cells induced by seracertib (0μM~30μM). Gradient analysis was performed from 0 to 30 hours after treatment. Cell indices were normalized to the treatment time. Mean ± SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 17B] (A&B) This figure shows the real-time proliferation of LNCaP WT(E) and P1(F) cells induced by seracertib (0μM~30μM). Gradient analysis was performed from 0 to 30 hours after treatment. Cell indices were normalized to the treatment time. Mean ± SEM, N=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 18A] This figure shows the effect of PDE4D7 expression on cAMP dynamics within LNCaP. Analysis of the FRET ratio (YFP / CFP) in the cytosol of WT(A) or P1(C)LNCaP, or in the membrane of WT(B) or P1(D)LNCaP, after treatment with Rolipram or a combination of forskolin and IBMX. Mean + / - SEM, N=3, **p<0.01, ***p<0.001. [Figure 18B] This figure shows the effect of PDE4D7 expression on cAMP dynamics within LNCaP. Analysis of the FRET ratio (YFP / CFP) in the cytosol of WT(A) or P1(C)LNCaP, or in the membrane of WT(B) or P1(D)LNCaP, after treatment with Rolipram or a combination of forskolin and IBMX. Mean + / - SEM, N=3, **p<0.01, ***p<0.001. [Figure 18C] This figure shows the effect of PDE4D7 expression on cAMP dynamics within LNCaP. Analysis of the FRET ratio (YFP / CFP) in the cytosol of WT(A) or P1(C)LNCaP, or in the membrane of WT(B) or P1(D)LNCaP, after treatment with Rolipram or a combination of forskolin and IBMX. Mean + / - SEM, N=3, **p<0.01, ***p<0.001. [Figure 18D]This figure shows the effect of PDE4D7 expression on cAMP dynamics within LNCaP. Analysis of the FRET ratio (YFP / CFP) in the cytosol of WT(A) or P1(C)LNCaP, or in the membrane of WT(B) or P1(D)LNCaP, after treatment with Rolipram or a combination of forskolin and IBMX. Mean + / - SEM, N=3, **p<0.01, ***p<0.001. [Figure 18E] This figure shows the effect of PDE4D7 expression on cAMP dynamics within LNCaP. (E&F) Real-time proliferation analysis of WT(G) and P1(H) cells after treatment with the PDE4 activator MR-L2 (1~10 μM). Gradient analysis from 0 to 48 hours after treatment. Mean + / - SEM, N=3, **p<0.01, ***p<0.001. [Figure 18F] This figure shows the effect of PDE4D7 expression on cAMP dynamics within LNCaP. (E&F) Real-time proliferation analysis of WT(G) and P1(H) cells after treatment with the PDE4 activator MR-L2 (1~10 μM). Gradient analysis from 0 to 48 hours after treatment. Mean + / - SEM, N=3, **p<0.01, ***p<0.001. [Modes for carrying out the invention]

[0016] definition Although the present invention is described in relation to specific embodiments, this description should not be construed as restrictive.

[0017] Before describing in detail exemplary embodiments of the present invention, some definitions essential for understanding the invention are given.

[0018] As used herein and in the appended claims, the singular form includes its respective plural form unless the context explicitly states otherwise.

[0019] In the context of the present invention, the terms “about” and “approximately” refer to a range of accuracy in which a person skilled in the art will understand that the technical effect of the characteristics of the problem is still guaranteed. The terms typically indicate a deviation of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5% from the given numerical value.

[0020] "Approximately" and "about": When these terms refer to measurable values ​​such as quantities, time periods, and the like, the variation is intended to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, such that it is appropriate for carrying out the disclosed method.

[0021] "Antagonist" and "Inhibitor": These terms are used interchangeably and refer to compounds or agents that have the ability to reduce or inhibit the biological function of a target protein or polypeptide, for example, by reducing or inhibiting the activity or expression of the target protein or polypeptide. Therefore, the terms "antagonist" and "inhibitor" are defined in the context of the biological role of the target protein or polypeptide. Inhibitors do not need to completely suppress the biological function of the target protein or polypeptide, and in some embodiments, they reduce the activity by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. Some antagonists, as used herein, interact specifically with the target (e.g., specifically bind to the target), but compounds that inhibit the biological activity of the target protein or polypeptide by interacting with other members of the signaling pathway of the target protein or polypeptide are also specifically included in this definition. Non-limiting examples of biological activity inhibited by antagonists include biological activity related to tumor development, growth, or expansion, or undesirable immune responses that appear in autoimmune diseases.

[0022] "Anti-cancer effect": This refers to the effect a therapeutic agent has on cancer, such as reducing the proliferation, survival rate, or both of cancer cells. The IC50 of cancer cells can be used as a measure of anti-cancer effect. IC50 is a measure of the effectiveness of a therapeutic agent in inhibiting cancer cells by 50%.

[0023] "Reduce cancer": This term, in the context of specific cancers and / or their pathologies, refers to breaking down a tumor, for example, destroying the structural integrity or connective tissue of the tumor, resulting in a reduction in tumor size compared to the pre-treatment tumor size. "Reduce" cancer metastasis includes reducing the rate at which cancer spreads to other organs.

[0024] "Composition," "Product," or "Combination": These encompass compositions suitable for various routes of administration, including but not limited to intravenous, subcutaneous, intradermal, subdermal, intranodal, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol, and / or parenteral or mucosal application. Compositions, formulations, and products according to the inventions of this disclosure typically comprise a drug / compound / inhibitor (alone or in combination) and one or more suitable pharmaceutically acceptable excipients or carriers.

[0025] "Combination therapy," or "in combination with...": These terms refer to the use of one or more compounds or agents to treat a particular disorder or condition. For example, compound 1 may be administered in combination with at least one further therapeutic agent. The phrase "in combination with..." is not intended to imply that the other therapies and compound 1 must be administered simultaneously and / or formulated to be delivered together, although such delivery methods are within the scope of this disclosure. Compound 1 can be administered in parallel with, or before, one or more further agents (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks prior to, or after, one or more further agents (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks later). Generally, each therapeutic agent is administered in the dose and / or schedule determined for that particular agent. Other therapeutic agents can be administered together with Compound 1 as described herein, as a single composition, or separately as different compositions. Larger combinations, such as triple therapy, are also discussed herein.

[0026] It should be understood that the term "including" is not restrictive. For the purposes of this invention, the term "consisting of" is considered a preferred embodiment of the term "including." Hereinafter, when a group is defined as containing at least a certain number of embodiments, this is intended to also preferably include groups consisting only of these embodiments.

[0027] Furthermore, terms such as “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” and similar terms in this specification and claims are used to distinguish similar elements from each other and are not necessarily intended to indicate a sequential or chronological order. It should be understood that such terms are interchangeable in appropriate contexts and that embodiments of the invention described herein can be operated in an order other than those described or illustrated herein.

[0028] Where terms such as “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d)” relate to a method or step of use, unless otherwise specified in this application as described above or below, there may be no time or period coherence between steps, i.e., the steps may be performed simultaneously, or there may be periods of seconds, minutes, hours, days, weeks, months or even years between such steps.

[0029] As used herein, the term “at least” means a specific value greater than or equal to that specific value. For example, “at least 2” is understood to be the same as “2 or greater,” i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15… and so on. As used herein, the term “maximum” means a specific value less than or equal to that specific value. For example, “maximum 5” is understood to be the same as “5 or less,” i.e., 5, 4, 3,…, -10, -11… and so on.

[0030] As used herein, the word "includes" or its variations, such as "containing," is understood to include the specified element, number, or step, or group of elements, numbers, or steps, but not to exclude any other element, number, or step, or group of elements, numbers, or steps. The verb "includes" includes the verbs "essentially consist of" and "consist of."

[0031] As used herein, the term “conventional techniques” refers to situations in which the methods used in the methods of the present invention are apparent to those skilled in the art. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing, and related fields is well known to those skilled in the art and is discussed, for example, in the following references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodically updated; and the series Methods in Enzymology, Academic Press, San Diego.

[0032] As used herein, the term “identity” refers to a measure of the identity of a nucleotide or amino acid sequence. Generally, sequences are aligned to obtain the greatest order fit. “Identity” has a meaning recognized in the art by itself and can be calculated using published techniques. See, for example: (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics And Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis Of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis In Molecular Biology, Von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer; Gribskov, M. and Develeux, J., eds., M Stockton Press, New York, 1991). While several methods exist for determining the identity between two nucleotide or amino acid sequences, the term "identity" is well known to those skilled in the art (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073). Methods commonly used to determine the identity or similarity between two sequences include, but are not limited to, those disclosed in Guide To Huge Computers, edited by Martin J. Bishop, Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., Siam J. Applied Math (1988) 48:1073. Methods for determining identity and similarity have been systematized in computer programs.Preferred computer programming methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, and FASTA (Atschul, SF et al., J. Molec. Biol. (1990) 215:403).

[0033] As an example, a polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence encoding a particular polypeptide sequence is intended to have a nucleotide sequence identical to the reference sequence, except that the polynucleotide sequence may contain up to five point mutations for every 100 nucleotides of the reference amino acid sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted and / or replaced with other nucleotides, and / or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence occur at the 5' or 3' terminal position of the reference nucleotide sequence, or between those terminal positions, individually between nucleotides in the reference sequence, or scattered within the reference sequence as one or more adjacent groups.

[0034] Similarly, by having a polypeptide having an amino acid sequence that is at least, for example, 95% "identical" to the reference amino acid sequence of SEQ ID NO: X, the amino acid sequence of the polypeptide is intended to be identical to the reference sequence, except that the amino acid sequence may contain up to five amino acid changes for every 100 amino acids of the reference amino acid sequence of SEQ ID NO: X. In other words, in order to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with other amino acids, or up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These changes in the reference sequence occur at the amino or carboxyl terminal positions of the reference amino acid sequence, or between those terminal positions, individually between residues in the reference sequence, or scattered as one or more adjacent groups within the reference sequence.

[0035] As used herein, the term "in vitro" refers to an experimental method or measurement performed using components of an organism isolated from its natural state.

[0036] As used herein, the term “ex vivo” refers to an experimental method or measurement performed in or on a living tissue in an external environment that has minimal alteration to its natural state.

[0037] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are intended to include DNA molecules and RNA molecules. Nucleic acids (molecules) may be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0038] As used herein, the term “sequence” refers to a nucleotide, or, when referring to a “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence,” it refers to the sequence of nucleotides in or within a nucleic acid and / or polynucleotide. In the context of the present invention, the first nucleic acid sequence may be contained within a further nucleic acid sequence and may overlap with a further nucleic acid sequence.

[0039] As used herein, the terms “subject,” “individual,” “animal,” “patient,” or “mammal” are interchangeable and refer to any subject, particularly mammalian subjects, for which diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, livestock, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, bears, etc. Where provided herein, subjects may be alive or dead. Samples may be taken post-mortem, i.e., from a subject after death, and / or samples may be taken from a living subject.

[0040] As used herein, the terms “treat,” “treat,” “alleviate,” “reduce,” or “relieve” refer to methods for achieving beneficial or desirable outcomes, including but not limited to therapeutic benefits. The therapeutic benefit is intended to be the eradication, remission, or reduction (or delay) of the progression of the underlying disease being treated. Furthermore, the therapeutic benefit is achieved by the eradication, remission, or reduction (or delay) of the progression of one or more physiological symptoms associated with the underlying disease, such that the patient may still be affected by the underlying disease, but an improvement, delay, or reduction in the progression of one or more of those symptoms is observed in the patient.

[0041] Detailed description of the embodiment When used herein, section headings are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0042] Parts of this invention contain copyrighted material (for example, but not limited to, figures, photographs of the apparatus, or any other aspects of this proposal, with respect to which copyright protection is or may be available in any jurisdiction). The copyright holder has no objection to any reproduction of the patent document or patent invention by any person in accordance with the patent files or records of the Patent Office, but otherwise retains all copyrights.

[0043] Various terms relating to the methods, compositions, uses, and other embodiments of the present invention are used throughout the specification and claims. Unless otherwise indicated, such terms should be given their common meanings in the art to which the present invention relates. Other specifically defined terms should be interpreted in accordance with the definitions provided herein. Preferred materials and methods are described herein, but any similar or equivalent methods and materials may be used in the execution of the tests of the present invention.

[0044] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art.

[0045] Activation of G protein-coupled receptors and subsequent activation of adenyl cyclase catalyzes the conversion of ATP to cAMP, a second messenger involved in regulating numerous cellular processes. Phosphodiesterases (PDEs) are a superfamily of enzymes that play a fundamental role in hydrolyzing cAMP, leading to its degradation and the termination of cellular signaling. These enzymes regulate and control the intracellular cAMP gradient around specific signaling complexes, with each specific isoform having its own unique functional role.

[0046] The PDE4 family of enzymes is particularly problematic, comprising over 25 different isoforms, many of which possess important, non-overlapping functions. Often, the function of a particular PDE4 isoform is handed down by its unique N-terminus, which acts as a "zip code" to retain the PDE4 enzyme in its isolated intracellular domain, where it forms a signal-specific cAMP gradient. PDE4 also contains catalytic units and regulatory domains called "upstream conserved regions 1 and 2" (UCR1 / 2), which are highly conserved across the isoforms. All long forms of PDE4, e.g., PDE4D7, contain UCR1, which contains a PKA motif that becomes phosphorylated under cAMP-elevating conditions. Such action functions to activate PDE4, rapidly reducing local cAMP concentrations. This feedback loop underscores the transient nature of the cAMP signaling, ensuring a rapid but instantaneous response to Gαs-coupled receptor activation.

[0047] Deregulated cAMP signaling, along with the PDE4D subfamily, which is particularly problematic in prostate cancer (PCa), is associated with a range of diseases, including cancer. PDE4D7 is a long PDE4D isoform variant that localizes submembranely in PCa cells, and its expression is inversely correlated with disease progression. In particular, PDE4D7 mRNA is significantly downregulated from androgen-sensitive (AS) to androgen-insensitive (AI) disease phenotypes, indicating the involvement of PDE4D7 in PCa development and progression. Furthermore, there is a strong correlation between PDE4D7 expression and the presence of TMPRSS2-ERG gene fusion, which is the most common gene rearrangement in PCa and leads to ERG overexpression and subsequent enhancement of PCa malignancy.

[0048] This specification describes data obtained from prostate cancer cells in which PDE4D7 expression was particularly reduced using shRNA-mediated knockdown. Surprisingly, we found that re-expression of PDE4D7 in these knockdown cells reduced proliferation. Furthermore, PDE4D7 expression was able to reduce resistance to drug therapy, such as resistance to antiandrogens, PARP inhibitors, or chemotherapy. Based on these data, we concluded that increasing PDE4D7 expression or increasing PDE4D7 phosphodiesterase activity can reduce therapy resistance and may be useful in treating therapy-resistant prostate cancer.

[0049] Therefore, in a first embodiment, the present invention relates to a product for use in the treatment, prevention or remission of prostate cancer by reducing therapy resistance in subjects requiring such treatment, wherein the product induces the expression of PDE4D7 or promotes the activity of phosphodiesterase 4D7. Alternatively, the present invention relates to a method for treating, preventing or remitting prostate cancer in subjects requiring such treatment, wherein the method comprises the step of administering to a subject a product that induces the expression of PDE4D7 or promotes the activity of phosphodiesterase 4D7 and is selected from the following: A polynucleotide encoding a peptide having the sequence of SEQ ID NO: 2, or a variant thereof, wherein the variant is a polynucleotide encoding a peptide having a sequence having at least 90% sequence homology to SEQ ID NO: 2, and the peptide has phosphodiesterase activity, or A polynucleotide comprising the nucleotide sequence defined in SEQ ID NO: 1, or a variant thereof, wherein the variant comprises a nucleotide sequence having at least 90% sequence homology to SEQ ID NO: 1, and the polynucleotide is a variant encoding a peptide having phosphodiesterase activity, or A peptide comprising the peptide sequence defined in SEQ ID NO: 2, or a variant thereof, wherein the variant comprises a peptide sequence having at least 90% sequence homology to SEQ ID NO: 2, and the peptide has phosphodiesterase activity, or A peptide comprising a peptide sequence encoded by a polynucleotide comprising the nucleotide sequence defined in SEQ ID NO: 1, or a variant thereof, wherein the variant comprises a peptide sequence encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence homology to SEQ ID NO: 1, and the polynucleotide encodes a peptide having phosphodiesterase activity, or A compound defined by formula I, [ka] During the ceremony, R1 is H, (C1-4) alkyl, or (C1-4) alkyloxy. R2 and R6 are selected independently of H. R3, R4, and R5 are H, halogen, CN, (C 1~4 ) Alkyl and (C 1~4 ) Independently selected from alkyloxy, and the (C 1~4 ) Alkyl and (C 1~4 The alkyloxy group may be substituted with 1 to 3 fluorocarbons. R7, R8, and R 10 It is selected independently from H and F, R9 is selected from (C1-4) alkyl, (C1-4) alkyloxy, CN, and halogen, and the (C1-4) alkyl and (C1-4) alkyloxy groups may be substituted with 1-3 fluorocarbons, in the compound. or A polynucleotide encoding a peptide containing a sequence selected from SEQ ID NOs: 24, 25, 26, or 27, or a sequence consisting of a sequence selected from SEQ ID NOs: 24, 25, 26, or 27, or a peptide containing a sequence selected from SEQ ID NOs: 24, 25, 26, or 27, or a sequence consisting of a sequence selected from SEQ ID NOs: 24, 25, 26, or 27.

[0050] As previously shown, the present invention relates in one aspect to phosphodiesterase 4D7 (PDE4D7) for use as a therapeutic strategy. The terms “phosphodiesterase 4D7” or “PDE4D7” refer to the splice variant 7 of human phosphodiesterase PDE4D, i.e., the human phosphodiesterase PDE4D7 gene, preferably the sequence defined by Genbank accession number AF536976 (type AF536976.1, GI:22901883 as of March 3, 2009), more preferably the nucleotide sequence shown in SEQ ID NO: 1, corresponding to the previously shown Genbank accession number of the PDE4D7 transcript and the sequence of the corresponding protein accession number AAN10118 (type AAN10118.1), and also refer to the corresponding amino acid sequence shown in SEQ ID NO: 2, corresponding to the previously shown Genbank accession number of the PDE4D7 polypeptide encoded by the PDE4D7 transcript. This term refers to a nucleotide sequence that shows a high degree of homology to PDE4D7, for example, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 1, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 2 This also includes nucleic acid sequences encoding amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence or the sequence shown in Sequence ID No. 2, or amino acid sequences encoded by nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in Sequence ID No. 1.

[0051] The terms “human phosphodiesterase PDE4D7 gene,” “PDE4D7 gene,” or “PDE4D7 marker gene,” as used herein, refer to the gene encoding phosphodiesterase 4D. Preferably, the term refers to a gene that expresses phosphodiesterase 4D as a specific exon combination defined by splice variant 7, for example, Genbank accession number AF536976 (type AF536976.1, GI:22901883 as of March 3, 2009), or the gene shown in Sequence ID No. 1. The term also refers to a DNA molecule, preferably a cDNA molecule, derived from an mRNA transcript encoding phosphodiesterase 4D, spliced ​​as variant 7.

[0052] The terms “phosphodiesterase 4D7,” “phosphodiesterase 4D7 protein,” or “PDE4D7 protein,” as used herein, refer to the protein encoded by the 4D7 splice variant as previously defined herein. Preferably, the term refers to the protein encoded by the phosphodiesterase 4D gene as a specific exon combination defined by splice variant 7, for example, Genbank accession number AF536976 (type AF536976.1, GI:22901883 as of March 3, 2009), or the protein shown in Sequence ID No. 2. The term also refers to proteins or polypeptides derived from the protein encoded by the PDE4D7 gene that has been modified by the addition of further amino acids, or by other modifications, such as sugar residues, tags, phosphorylation, acetylation, methylation, ubiquitination, or other modifications, or by the removal of one or more amino acids, such as signal peptides, but not limited to those listed below.

[0053] This invention is facilitated by the inventors' findings that reintroducing PDE4D7 into PDE4D7 knockdown prostate cancer cells has several beneficial effects, such as reducing cancer cell proliferation and restoring therapy resistance. Such effects are expected to be established in two ways, as follows: 1) by increasing the expression of the PDE4D7 protein, i.e., by increasing the amount of phosphodiesterase 4D7 protein in prostate cancer cells, or indirectly by increasing the expression level of the PDE4D7 isoform of the PDE4D gene, i.e., by promoting the transcription of PDE4D gene isoform 7, or otherwise by increasing the amount of PDE4D7 mRNA; or 2) by increasing the activity of the PDE4D7 enzyme, for example by removing or inhibiting the antagonist of the PDE4D7 protein enzymatic activity, or by stimulating the agonist of the PDE4D7 protein enzymatic activity, or by introducing the active form of the PDE4D7 protein by introducing further proteins (directly or indirectly), or by compounds that directly or indirectly involve and activate PDE4D7.

[0054] Therefore, this disclosure broadly defines products for inducing the expression of PDE4D7 or promoting the activity of phosphodiesterase 4D7. In one embodiment, the product is a compound or a composition.

[0055] As used herein, the term "compound" refers to a chemical compound (also known as a "small molecule therapy"), a polynucleotide, a polypeptide, or a glycopolymer, and is not limited to these biological compounds.

[0056] As used herein, the term "composition" refers to a mixture of compounds or a combination of compounds and auxiliaries. A composition may be a solution or a dry composition.

[0057] Several methods for achieving increased PDE4D7 expression or phosphodiesterase 4D7 activity in subjects are envisioned, and several non-limiting examples are listed below. However, it should be understood that this disclosure extends to any method or compound known to those skilled in the art for achieving increased PDE4D7 expression or increased activity of phosphodiesterase 4D7 isoforms. Therefore, in one embodiment of this disclosure, the product is selected from the following: - Gene therapy to induce PDE4D7 expression, - Compounds that induce PDE4D7 expression, - Compounds that directly stimulate or promote phosphodiesterase 4D7 activity, - Compounds that indirectly stimulate or promote phosphodiesterase 4D7 activity, - Inhibitors of PDE4D7 transcription inhibitors, - Inhibitors of phosphodiesterase 4D7 enzyme activity - mRNA encoding phosphodiesterase 4D7 protein, - Phosphodiesterase 4D7 protein, - An amyloid-beta peptide that promotes phosphodiesterase 4D7 activity.

[0058] As part of the present invention, the following products are assumed to increase the expression or activity of PDE4D7: 1) Directly introducing a protein having PDE4D7 activity. Therefore, in one embodiment, the present invention describes a polynucleotide or variant thereof encoding a peptide having the sequence of SEQ ID NO: 2, wherein the variant is a polynucleotide encoding a peptide having a sequence having at least 90% sequence homology to SEQ ID NO: 2, and the peptide has phosphodiesterase activity. SEQ ID NO: 2 describes the amino acid sequence of PDE4D isoform 7. Therefore, a peptide (or a variant thereof as defined herein) containing or derived from the amino acid sequence defined by SEQ ID NO: 2 can be directly introduced into cells. Methods for introducing peptides into cells are known to those skilled in the art and include, but are not limited to, the use of cell-permeable peptides and nanoparticles. 2) Introducing a polynucleotide encoding a protein having PDE4D7 activity. Therefore, in one embodiment, the present invention describes a polynucleotide or a variant thereof comprising the nucleotide sequence defined in SEQ ID NO: 1, wherein the variant is a polynucleotide comprising a nucleotide sequence having at least 90% sequence homology to SEQ ID NO: 1, and the polynucleotide encodes a peptide having phosphodiesterase activity. 3) A compound defined by formula I, [ka] During the ceremony, R1 is H, (C1-4) alkyl, or (C1-4) alkyloxy. R2 and R6 are selected independently of H. R3, R4, and R5 are H, halogen, CN, (C 1~4 ) Alkyl and (C 1~4 ) Independently selected from alkyloxy, and the (C 1~4 ) Alkyl and (C 1~4 The alkyloxy group may be substituted with 1 to 3 fluorocarbons. R7, R8, and R 10 It is selected independently from H and F, A compound in which R9 is selected from (C1-4) alkyl, (C1-4) alkyloxy, CN, and halogen, and the (C1-4) alkyl and (C1-4) alkyloxy groups may be substituted with 1-3 fluorocarbons. 4) A peptide having a sequence that includes a sequence selected from SEQ ID NOs: 24, 25, 26, or 27, or a sequence consisting of a sequence selected from SEQ ID NOs: 24, 25, 26, or 27, or a polynucleotide encoding a peptide that includes a sequence selected from SEQ ID NOs: 24, 25, 26, or 27, or a sequence consisting of a sequence selected from SEQ ID NOs: 24, 25, 26, or 27.

[0059] As used herein, gene therapy for inducing PDE4D7 expression refers to a method for introducing a nucleotide encoding a phosphodiesterase 4D7 isoform into cells, preferably cells of a subject with prostate cancer, more preferably prostate cancer cells or their metastases. The nucleotide may be, for example, a viral vector or a non-viral vector, but it is understood that any nucleotide that can be introduced into the patient's cells and express PDE4D7 may be used. For example, the nucleotide may be mRNA encoding PDE4D7 or a variant of PDE4D7. The nucleotide, such as mRNA, is expressed in vivo in tumor cells using lipid nanoparticles (LNPs). Suitable LNP formulations are known to those skilled in the art.

[0060] Viral vectors are a known strategy for gene therapy, and typically, viral vector gene therapy uses a modified virus as a drug delivery medium to introduce a specific DNA or RNA sequence into cells. The vector is packaged using viral proteins, which enable the infection of cells and the expression of their viral genome. Viral vectors are typically modified so as not to generate new viral particles upon infection of target cells. Non-limiting examples include retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV), lentiviruses (LV), and herpes simplex virus (HSV). Other forms of using viral particles or viral vectors to introduce the nucleotide encoding PDE4D7 are known to those skilled in the art and are also intended to be included in the present invention.

[0061] Non-viral vectors may be used instead of viral vector-based gene therapy. These vectors typically contain promoters to drive the expression of the relevant construct (e.g., PDE4D7) and usually require several means to introduce the vector into target cells. Means for delivering non-viral vectors to target cells in subjects are known to those skilled in the art and are evaluated, for example, in Ramamoorth M, Narvekar A. Non-viral vectors in gene therapy - an overview. J Clin Diagn Res. 2015 / 1;9(1):GE01-6 (which is incorporated herein by reference in its entirety). For example, nanoparticles can be used to encapsulate vectors. Non-limiting examples include lipid-based nanoparticles, peptide-based nanoparticles, cationic lipid-based nanoparticles, apolipoprotein-based nanoparticles, and (synthetic) polymer-based nanoparticles, such as polyethyleneimine (PEI), chitosan, polylactic acid, polylactide, polyglucoside, dendrimer, or polymethacrylate-based nanoparticles. As used herein, nanoparticles are small particles that can be used as carriers for delivering a cargo (package) in a patient. Preferably, the cargo is a product as broadly defined herein. Thus, nanoparticles can be used to deliver a product that induces the expression of PDE4D7 or promotes the activity of phosphodiesterase 4D7 to a site in a patient, such as a cell, tissue, or organ. Other forms of using nonviral vectors to introduce the nucleotide encoding PDE4D7 are known to those skilled in the art and are also envisioned to be included in the present invention.

[0062] Therefore, in one embodiment, the gene therapy is selected from a viral vector capable of expressing PDE4D7 in the subject, or a non-viral vector capable of expressing PDE4D7 in the subject. In one embodiment, the product is delivered using nanoparticles.

[0063] As used herein, the term vector is used to refer to any particle (e.g., plasmid, cosmid, lambda phage) used as a medium for artificially transporting an exogenous nuclear sequence—usually DNA—into another cell, where the exogenous nuclear sequence is replicated and / or expressed.

[0064] As used herein, the term "compound that induces PDE4D7 expression" is intended to refer to any biological or chemical compound that can increase the expression of PDE4D7. This is achieved, for example, by promoting the transcription of the PDE4D7 gene or by inhibiting the degradation of the phosphodiesterase 4D7 isoform protein. For example, a compound may indirectly promote the transcription of PDE4D7 by participating in a signaling pathway, or it may directly interact with the promoter region of genomic DNA to promote the transcription of the PDE4D7 isoform.

[0065] As used herein, promoting the transcription of PDE4D7 means increasing the transcription of PDE4D7 to result in a greater amount of PDE4D7 mRNA transcript and / or preferably a greater amount of phosphodiesterase 4D7 isoform protein in the cell. The promotion may be specific to PDE4D7, specific to all PDE4D isoforms, specific to all PDE4, and even specific to all PDE family members. In other words, increasing the expression of PDE4D7 does not preclude increased expression of other PDE4D isoforms, other PDE4 family members, and even other PDE family members.

[0066] As used herein, compounds that directly stimulate or promote phosphodiesterase 4D7 activity are intended to refer to compounds directly involved in the enzymatic activity of the phosphodiesterase 4D7 isoform protein. While we do not wish to be bound by theory, it is theorized that PDE proteins, such as the PDE4D7 protein, can exist in active and inactive structures, and that activity may be induced by other compounds through interactions with the PDE protein, for example, by promoting the protein's active structure, or by blocking or preventing the binding of inhibitors, or by modifying the protein to promote activity (e.g., by phosphorylation or other known protein modifications). As used herein, enzymatic activity, when referring to phosphodiesterase, refers to the catalytic activity of the hydrolysis of cAMP and / or cGMP.

[0067] As used herein, compounds that indirectly stimulate or promote phosphodiesterase 4D7 activity are intended to refer to compounds that indirectly participate in the enzymatic activity of the phosphodiesterase 4D7 isoform protein. As above, it is assumed that a compound induces an activator of PDE4D7 or blocks an inhibitor of PDE4D7 activity, and thus indirectly affects the enzymatic activity of the protein. Those skilled in the art are familiar with methods for determining PDE or, in particular, PDE4D7 activity. For example, commercially available assays for PDE activity are available, and a method is described, for example, in Blair et al. Measuring cAMP Specific Phosphodiesterase Activity: A Two-step Radioassay. Bio Protoc. 2020 / 4 / 5;10(7):e3581, which is incorporated herein by reference throughout.

[0068] As used herein, a PDE4D7 transcription inhibitor refers to a compound capable of blocking an inhibitor of PDE4D7 gene transcription. A PDE4D7 gene transcription inhibitor is either a direct inhibitor that binds to genomic DNA and prevents or reduces gene transcription, or an indirect inhibitor that reduces or inhibits PDE4D7 gene transcription through downstream action.

[0069] As used herein, an inhibitor of a phosphodiesterase 4D7 enzyme activity inhibitor refers to a compound capable of blocking an inhibitor of phosphodiesterase isoform 4D7 protein activity. For example, the compound functions by degrading the inhibitor, preventing the inhibitor from engaging with PDE4D7, or inhibiting the activity of the inhibitor.

[0070] As used herein, mRNA encoding the phosphodiesterase 4D7 protein refers to an RNA molecule that can be translated into the protein corresponding to SEQ ID NO: 2 or a substantially similar protein. mRNA molecules generally include untranslated elements, e.g., the 5' and 3' UTRs. Direct introduction of PDE4D7 mRNA into target cells (e.g., prostate cancer cells in a subject) is expected to upregulate PDE4D7. Methods for delivering mRNA to target cells are known to those skilled in the art, for example, by using the nanobodies described above.

[0071] As used herein, phosphodiesterase 4D7 protein refers to a compound or biosimilar containing the PDE4D7 protein, or a constitutively active variant thereof having phosphodiesterase activity. Direct introduction of the PDE4D7 protein into target cells (e.g., prostate cancer cells in a subject) is expected to increase PDE4D7 activity. Methods for delivering the protein to target cells are known to those skilled in the art, for example, by using the nanobodies described above.

[0072] As used herein, a peptide that promotes phosphodiesterase 4D7 activity refers to a peptide capable of increasing the activity of the PDE4D7 enzyme. A non-limiting example is the amyloid-beta (Abeta) peptide, which has been shown to particularly increase the activity of long PDE4D isoforms such as PDE4D7. As used herein, amyloid-beta, also known as Aβ or Abeta, refers to a 37-49 amino acid peptide that is the main component of amyloid plaques found in the brains of people with Alzheimer's disease (Chen et al. Acta Pharmacol Sin 38, 1205-1235 (2017)). Amyloid-beta peptide (Aβ) is produced via the proteolytic processing of the transmembrane protein amyloid precursor protein (APP) by β- and γ-secretases.

[0073] Therefore, peptides that promote phosphodiesterase 4D7 activity are preferably Abeta peptides or derivatives thereof. As used herein, Abeta peptides or derivatives thereof are characterized as peptides having a length of 16 to 50 amino acids and containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence defined by SEQ ID NO: 24 (Abeta Core Peptide 1), and more preferably containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence defined by SEQ ID NO: 25 (Abeta Core Peptide 2). Non-limiting examples are shown by Abeta42 (SEQ ID NO: 26) and Abeta40 (SEQ ID NO: 27). Therefore, in one embodiment, the Abeta peptide or its derivative is characterized as a peptide having a length of 42 to 50 amino acids and containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence defined by SEQ ID NO: 26, or a peptide having a length of 40 to 50 amino acids and containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence defined by SEQ ID NO: 27. Sequence ID No. 24 Abeta Core Peptide 2 HDSGYEVHHQKLVFFAEDVGSNKGAIIG Sequence ID No. 25 Abeta Core Peptide 2 FRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMV Sequence ID 26: Abeta42 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA Sequence ID 27 Abeta40 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV

[0074] This specification describes several advantages of therapies based on increasing PDE4D7 expression or activating phosphodiesterase 4D7 in cells, such as reducing cancer cell proliferation. Furthermore, experimental data strongly suggest that increasing PDE4D7 expression or activating phosphodiesterase 4D7 in cells helps overcome therapy resistance, or at least makes therapy-resistant tumor cells more responsive to therapeutic agents, such as anti-androgens, PARP inhibitors, and taxanes. Thus, in one embodiment, treatment, prevention, or remission includes reducing therapy resistance.

[0075] As used herein, reducing therapy resistance means reducing or avoiding resistance to the therapy that has occurred or may occur in cancer cells. As a result, when a compound (product) that induces PDE4D7 expression or promotes phosphodiesterase 4D7 activity is administered in conjunction with a therapy, the efficacy of the therapy is unexpectedly increased, in addition to the beneficial effects observed from the compounds described herein.

[0076] Therefore, in one embodiment, therapy resistance is resistance to antiandrogen drugs.

[0077] Antiandrogens may be androgen receptor antagonists. Non-limiting examples of steroidal antiandrogens include 17α-hydroxyprogesterone derivatives, e.g., chlormadinone acetate, cyproterone acetate, megestrol acetate, osaterone acetate; 19-norprogesterone derivatives, e.g., nomegestrol acetate; 19-nortestosterone derivatives, e.g., dienogest, oxendrone; 17α-spirolactone derivatives, e.g., drospirenone, spironolactone, and others, e.g., medrogestone. Non-limiting examples of nonsteroidal antiandrogens include bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide, proxalutamide, cimetidine, and topirutamide.

[0078] Antiandrogen drugs may also be androgen synthesis inhibitors. Non-limiting examples include CYP17A1 inhibitors, such as abiraterone acetate, ketoconazole, and ceviteronel; CYP11A1 (P450scc) inhibitors, such as aminoglutethimide; and 5α-reductase inhibitors, such as alpha-estradiol, dutasteride, epristeride, finasteride, and saw palmetto extract.

[0079] Antiandrogens may also be antigonadotropins. Non-limiting examples include estrogens, e.g., estradiol (and its esters), ethinylestradiol, conjugated estrogens, diethylstilbestrol; GnRH analogs, e.g., GnRH agonists such as goserelin and leuprorelin, GnRH antagonists such as cetrorelix; and progestogens, e.g., chlormadinone acetate, cyproterone acetate, gestolone caproate, medroxyprogesterone acetate, megestrol acetate.

[0080] Therefore, in one embodiment, therapy resistance is resistance to androgen receptor antagonists, androgen synthesis inhibitors, or antigonadotropins, preferably the androgen receptor antagonists, androgen synthesis inhibitors, or antigonadotropins listed above.

[0081] In a more preferred embodiment, therapy resistance is resistance to enzalutamide, flutamide, nilutamide, bicalutamide, topirutamide, apalutamide, darolutamide, cimetidine, ketoconazole, proxalutamide (GT-0918), ceviteronel (VT-464), or abiraterone. In a more preferred embodiment, therapy resistance is resistance to enzalutamide.

[0082] In one embodiment, the therapy resistance is PARP inhibitor resistance.

[0083] PARP inhibitor resistance may be resistance to olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib (BGB-290), rucaparib, CEP9722, E7016, iniparib, or 3-aminobenzamide. Therefore, in one embodiment, therapy resistance is resistance to the PARP inhibitors listed above. In one embodiment, therapy resistance is resistance to a PARP inhibitor selected from olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib (BGB-290), rucaparib, CEP9722, or E7016. In one embodiment, therapy resistance is resistance to olaparib.

[0084] In one embodiment, therapy resistance is defined as resistance to chemotherapeutic agents.

[0085] The chemotherapeutic agent may be an alkylating agent. Non-limiting examples of alkylating agents include cyclophosphamide, mechloretamine, chlorambucil, melphalan, dacarbazine, nitrosourea, and temozolomide. The chemotherapeutic agent may be an anthracycline. Non-limiting examples of anthracyclines include daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and barurubicin. The chemotherapeutic agent may be a taxane. Non-limiting examples of taxanes include paclitaxel, docetaxel, abraxane, and taxotere. The chemotherapeutic agent may be a histone deacetylase inhibitor. Non-limiting examples of histone deacetylase inhibitors include vorinostat and romidepsin. The chemotherapeutic agent may be a topoisomerase inhibitor. Non-limiting examples of topoisomerase inhibitors include irinotecan, topotecan, etoposide, teniposide, and tafluposide. The chemotherapeutic agent may also be a kinase inhibitor. Non-limiting examples of kinase inhibitors include bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and bismodegib. The chemotherapeutic agent may also be a nucleotide analog or a precursor analog. Non-limiting examples of nucleotide analogs or precursor analogs include azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine. The chemotherapeutic agent may also be a platinum-based agent. Non-limiting examples of platinum-based agents include carboplatin, cisplatin, and oxaliplatin. The chemotherapeutic agent may also be a retinoid. Non-exclusive examples of retinoids include tretinoin, alitretinoin, and bexarotene. Chemotherapy agents may be vinca alkaloids or derivatives. Non-exclusive examples of vinca alkaloids or derivatives include vinblastine, vincristine, vindesine, and vinorelbine.

[0086] Therefore, in one embodiment, therapy resistance is resistance to the chemotherapeutic agents listed above. In one embodiment, resistance to chemotherapeutic agents is resistance to paclitaxel, docetaxel, Abraxane, Taxotere, cabazitaxel, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, barrubicin, estramustine phosphate, allestramustine, atrimustine, cistethrol acetate, estradiol mustard, estramustine, estromustine, ICI-85966, or fenestrol. In one embodiment, resistance to chemotherapeutic agents is resistance to osetaxel, cabazitaxel, mitoxantrone, and estramustine.

[0087] The present invention further provides that a further therapy, preferably one in which resistance to the therapy is avoided or reversed by a product defined herein, is administered to a subject.

[0088] Therefore, in one embodiment, treatment, prevention, or remission further comprises administering an antiandrogenic agent. The antiandrogenic agent may be selected from the antiandrogenic agents described above. In one embodiment, treatment, prevention, or remission further comprises administering an antiandrogenic agent selected from enzalutamide, flutamide, nilutamide, bicalutamide, topirutamide, apalutamide, darolutamide, cimetidine, ketoconazole, proxalutamide (GT-0918), or ceviteronel (VT-464), or abiraterone. In one embodiment, treatment, prevention, or remission further comprises administering enzalutamide.

[0089] Therefore, treatment, prevention, or remission further comprises administering a PARP inhibitor. The PARP inhibitor may be selected from the PARP inhibitors described above. In one embodiment, treatment, prevention, or remission further comprises administering a PARP inhibitor selected from olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib (BGB-290), rucaparib, CEP9722, or E7016. In one embodiment, treatment, prevention, or remission further comprises administering olaparib.

[0090] Therefore, in one embodiment, treatment, prevention, or remission further comprises administering a chemotherapeutic agent. The chemotherapeutic agent may be one of the chemotherapeutic agents listed above. In one embodiment, treatment, prevention, or remission further comprises administering a chemotherapeutic agent selected from paclitaxel, docetaxel, Abraxane, Taxotere, cabazitaxel, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, barrubicin, estramustine phosphate, allestramustine, atrimustine, cistethrol acetate, estradiol mustard, estramustine, estromustine, ICI-85966, or fenestrol. In one embodiment, treatment, prevention, or remission further comprises administering a chemotherapeutic agent selected from osetaxel, cabazitaxel, mitoxantrone, and estramustine.

[0091] In one embodiment, the products broadly described herein are administered to a subject when the subject exhibits therapy resistance. For example, therapy resistance may be androgen depletion resistance or PARP inhibitor resistance. In such cases, PDE4D7 expression is reduced, and the subject is very likely to benefit from the products broadly described herein. In one embodiment, if a tumor derived from the subject has low PDE4D7 expression, in such cases, it is assumed that PDE4D7 expression is reduced, and the subject is very likely to benefit from the products broadly described herein, and therefore the products broadly described herein are administered to the subject.

[0092] It is further assumed that, prior to administering any of the products broadly described herein, a tumor or tissue sample from the patient is prepared and analyzed for PDE4D7 expression. Therefore, in one embodiment, the present invention relates to a product for use in the treatment, prevention or remission of prostate cancer in a subject requiring such treatment, wherein the product induces the expression of PDE4D7 or promotes the activity of phosphodiesterase 4D7, and the treatment, prevention or remission comprises preparing a sample from the subject and determining the expression level of PDE4D7. Alternatively, the present invention relates to a method for treating, preventing or relieving prostate cancer in a subject requiring such treatment, wherein the method is: - A step of preparing a sample derived from the aforementioned subject, - A step of determining the expression level of PDE4D7 in the sample, and - A step of administering a product as broadly defined herein to the subject. This relates to a method having

[0093] Instead of determining the expression level of PDE4D7, the enzymatic activity of the PDE4D7 protein may be determined. In one embodiment, if a low expression level of PDE4D7 is observed in the sample, the product is administered to the subject. In one embodiment, if a high expression level of PDE4D7 is observed in the sample, an alternative treatment (e.g., not one of the products specified herein) is administered to the subject. The alternative treatment may be, for example, a conventional treatment option for prostate cancer. Preferably, the alternative treatment in patients with localized disease is active surveillance (instead of aggressive treatment). Preferably, the alternative treatment in patients with non-localized disease is conventional / standard treatment or non-intensified treatment.

[0094] In one embodiment, the sample is a tumor sample or tissue sample derived from the subject, preferably a tumor sample, more preferably a prostate cancer tumor sample. For example, the tumor sample may be a biopsy or culture obtained from a tumor.

[0095] Those skilled in the art can easily determine PDE4D7 expression by, for example, preparing a reference library of prostate tumor samples, determining whether the expression level is considered "high" or "low," and can easily determine whether an overall range of PDE4D7-specific expression levels is observed in tumor samples, which values ​​correspond to high values ​​(e.g., above the median), and which values ​​correspond to low values ​​(e.g., below the median).

[0096] In an alternative embodiment of the present invention, a compound for use in the treatment, prevention, or remission of prostate cancer in a person requiring such use is provided, wherein the compound is an ATR inhibitor or a metabolic inhibitor. Preferably the compound is - When high expression of PDE4D7 is observed in tumors, - If low expression of PDE4D7 is observed, but PDE4D7 activity does not increase sufficiently to make the tumor susceptible again to inhibitor treatment, or - In the case of metastatic tumors It is administered to them.

[0097] The data presented herein in Examples 1–3 relating to ceraracertib (ATR inhibitor; see Figures 4D, 4E, and 17) and metformin (metabolism inhibitor; see Figures 6A and 6B) demonstrate that these inhibitors reduce tumor growth regardless of PDE4D7 expression levels. Tumors already exhibiting high levels of PDE4D7 may not respond to the products described more broadly. The same applies to metastatic tumors (compared to localized tumors) where increasing PDE4D7 activity is difficult. Furthermore, there may be tumors where increasing PDE4D7 activity is technically difficult for other reasons, for example, tumors that do not respond well to the products broadly defined herein. In these cases, the use of alternative therapies, such as ATR inhibitors or metabolism inhibitors, may be beneficial.

[0098] In one embodiment, the compound is an ATR inhibitor. In one embodiment, the ATR inhibitor is selected from RP-3500, seracertib (AZD6738), AZ20, elimusertib (BAY-1895344), elimusertib (BAY-1895344) hydrochloride, SKLB-197, ETP-46464, belzocertib (VE-822), dactricib (BEZ235), VE-821, trin 2, or CGK733. In a preferred embodiment, the ATR inhibitor is seracertib.

[0099] In one embodiment, the compound is a metabolic inhibitor. Suitable metabolic inhibitors are known to those skilled in the art, for example, as described in Lemberg et al., J Clin Invest. January 4, 2022; 132(1):e148550, and Luengo et al., Cell Chemical Biology 24, September 21, 2017, pp. 1161-1180. In one embodiment, the metabolic inhibitor is selected from methotrexate, pemetrexed, 6-mercaptopurine, 6-thioguanine, capecitabine, 5-fluorouracil, gemcitabine, cytarabine, leflunomide, CB-839, PEG-BCT-100 (ADIPEG20), AEB-1102, L-asparaginase, TVB-2640, AG-120 (ivosidenib), IDH305, BAY1436032, FT-2102, AG-221 (enasidenib), AG-881, AZD3965, CPI-613, or metformin. In a preferred embodiment, the metabolic inhibitor is metformin.

[0100] The present invention further provides a kit of elements comprising a product that induces the expression of PDE4D7 or promotes the activity of phosphodiesterase 4D7, as broadly defined herein. Thus, in one embodiment, the kit comprises a compound or composition that induces the expression of PDE4D7 or promotes the activity of phosphodiesterase 4D7. In one embodiment, the product is selected from the following: Gene therapy to induce PDE4D7 expression, Compounds that induce PDE4D7 expression, Compounds that directly stimulate or promote phosphodiesterase 4D7 activity, Compounds that indirectly stimulate or promote phosphodiesterase 4D7 activity, Inhibitors of PDE4D7 transcription inhibitors Inhibitors of phosphodiesterase 4D7 enzyme activity mRNA encoding phosphodiesterase 4D7 protein, Phosphodiesterase 4D7 protein, A peptide that promotes phosphodiesterase 4D7 activity.

[0101] In one embodiment, the gene therapy is selected from a viral vector capable of expressing PDE4D7 in the subject, or a non-viral vector capable of expressing PDE4D7 in the subject. In one embodiment, the product is contained in nanoparticles.

[0102] In one embodiment, the element kit further comprises an antiandrogenic drug and / or a PARP inhibitor and / or a chemotherapeutic agent. In one embodiment, the antiandrogenic drug is selected from enzalutamide, flutamide, nilutamide, bicalutamide, topirutamide, apalutamide, darolutamide, cimetidine, ketoconazole, proxalutamide (GT-0918), ceviteronel (VT-464), or abiraterone. In one embodiment, the PARP inhibitor is selected from olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib (BGB-290), rucaparib, CEP9722, or E7016. In one embodiment, the chemotherapeutic agent is selected from paclitaxel, docetaxel, abraxane, taxotere, cabazitaxel, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, barrubicin, estramustine phosphate, allestramustine, atrimustine, citestrol acetate, estradiol mustard, estramustine, estromustine, ICI-85966, or fenestrol.

[0103] In a further embodiment, the present invention relates to the ex vivo or in vitro use of products that induce PDE4D7 expression or promote phosphodiesterase 4D7 activity, as broadly defined herein, for increasing PDE4D7 expression and / or phosphodiesterase 4D7 activity in cells.

[0104] Any method, use, or composition described herein is considered applicable to any other method, use, or composition described herein. Embodiments discussed in the context of the methods, uses, and / or compositions of the present invention are applicable to any other method, use, or composition described herein. Thus, embodiments relating to one method, use, or composition are also applicable to other methods, uses, and compositions of the present invention. [Examples]

[0105] While the present invention has been described in general terms, it can be more readily understood by referring to the following examples, which are provided as illustrations and are not intended to be limitations of the present invention. [Examples]

[0106] method Production of stable, shRNA-transduced LNCaP cell lines Stable, shRNA-mediated, lentivirally transduced LNCaP cell lines were produced by AMSBIO (Table 1). Briefly, shRNA-PDE4D7 was cloned into a lentiviral vector and delivered to LNCaP cloned FGC cells (ATCC) via lentivirus (MOI=10). Cells were maintained in puromycin-supplemented medium before selection of stably transduced cells. Subsequently, stable shRNA-PDE4D7 LNCaP cell lines were retransduced with a lentivirus delivering PDE4D7 to produce retransduced cell lines. Scrambled shRNA was used as a control. All cells were cultured in RPMI1640 medium (Gibco) supplemented with 10% (v / v) fetal bovine serum, 1% (v / v) L-glutamine, and 1% (v / v) penicillin / streptomycin, and stored in a 37°C incubator with 5% CO2.

[0107] [Table 1]

[0108] Plasmid DNA transfection LNCaP WT and stable transducer cell lines were transiently transfected with the pcDNA3.1-PDE4D7-VSV plasmid DNA construct using Lipofectamine LTX (Thermo Fisher Scientific) with Plus reagent, according to the manufacturer's protocol. Cells were incubated for 24 hours post-transfection before further experiments.

[0109] xCELLigence Real-Time Cell Analysis (RTCA) Real-time cell proliferation was analyzed using the xCELLigence RTCA system (Agilent), which measures real-time cell adhesion across gold microelectrode biosensors in a 96-well electronic plate (E-plate) in a 37°C incubator with 5% CO2. Cell adhesion affects electrical impedance, which is converted to cell markers (CI) by RTCA software. Initial background measurements were recorded with 100 μL of RPMI1640 medium, followed by the addition of 100 μL (10,000 cells / well) of LNCaP cell suspension for measurement of CI over time. Approximately 24 hours after seeding, cells were treated with the indicated concentrations of drugs. Negative controls containing either 0.1% DMSO or untreated cells were also included. CI was converted to CI normalized to the treatment time using RTCA software.

[0110] Western blotting Cells were lysed in 3T3 lysis buffer (50 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 25 mM HEPES, 2.5 mM EDTA, 10% (v / v) glycerol, 1% (v / v) Triton X-100; pH 7.5) supplemented with cOmplete® EDTA-free protease inhibitor cocktail (Sigma-Aldrich). The lysed cells were rotated at 4°C for 30 minutes with inversion, and then centrifuged at 13,000 RPM at 4°C for 10 minutes. The supernatant was collected, and protein concentrations were determined by Bradford assay. Equivalent protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes for Western blotting. Before primary and secondary antibody incubation, the membranes were blocked with 5% (w / v) milk powder in TBS-T for 1 hour. Primary antibodies used: anti-PDE4D5 1:5000 (Baillie lab), anti-PDE4D7 1:500 (Baillie lab), anti-PDE4D9 1:5000 (Baillie lab), anti-Pan-PDE4D 1:5000 (Baillie lab), anti-VSV 1:5000 (Abcam, #ab1874), anti-GAPDH 1:5000 (Abcam, #ab8245), anti-AR 1:1000 (Abcam, #), anti-PSMA 1:1000, anti-E-cadherin 1:500 (Cell Signalling, #3195S), anti-TMPRSS2 1:1000 (Abcam#ab109131), anti-NKX3.1 1:1000 (Cell Signalling, #92998), anti-PSA 1:1000 (Abcam, #ab76113), anti-AR-V7 (Cell Signalling, #68492), anti-GR (Cell Signalling, #12041T).Secondary antibodies used: IRDye® 800CW donkey-anti-rabbit IgG 1:5000 (LI-COR, #926-32213), IRDye® 680RD donkey-anti-goat IgG 1:5000 (LI-COR, #926-68074), IRDye 800CW goat-anti-human IgG 1:5000 (LI-COR, #926-32232), IRDye® 680RD donkey-anti-mouse IgG 1:5000 (LI-COR, #925-68072).

[0111] RNA analysis qRT-PCR RNA was extracted using the RNeasy Mini kit (QIAGEN), and one-step qRT-PCR was performed using the Superscript® III Platinum® one-step qRT-PCR kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Experiments were performed using the Step-One® real-time PCR system (Thermo Fisher Scientific) with the following parameters: one cycle of 30 minutes at 50°C and 5 minutes at 95°C, followed by 45 cycles of 15 seconds at 95°C and 45 seconds at 60°C. Primers and probes were purchased from Integrated DNA Technologies at 400 nM primer / 200 nM probe and 1 ng / μl RNA per reaction (Table 2). Ct values ​​were quantified using the 2-ΔΔCt method, and statistical analysis was performed on the ΔΔCt values.

[0112] [Table 2]

[0113] NGS RNA sequencing Total RNA (100 ng) was extracted from LNCaP cell lines and analyzed by next-generation RNA sequencing (RNAseq) using a NovaSeq 6000 S4 system (2 x 150 bp read length). RNAseq reads were mapped to the human reference genome (Ensembl96) using STAR. Transcripts per million (TPM) values ​​were analyzed between WT and shRNA-transduced LNCaP cell lines and converted to z-scores. RNAseq data were further analyzed using iPathwayGuide (Advaita Bio) to calculate log-2x changes in genes associated with the prostate cancer pathway.

[0114] Immunofluorescence LNCaP cells were seeded on 13 mm coverslips 24 hours prior to the transfection described above. The cells were fixed with 4% (v / v) paraformaldehyde for 15 minutes, blocked, and permeabilized with 10% donkey serum, 0.5% BSA, and 0.2% Triton-X in PBS. The coverslips were incubated overnight with anti-PDE4D7 primary antibody (1:200, Baillie lab), followed by incubation in Alexa-Fluor488 donkey-anti-goat 1:500 secondary antibody (ThermoFisherScientific, #A-11055). The coverslips were placed on glass slides containing Duolink In Situ mounting medium with DAPI (Sigma-Aldrich) and imaged using a ZEISS LSM 880 laser scanning microscope with a 63x oil immersion objective lens.

[0115] statistical analysis Unless otherwise specified, values ​​are presented as mean ± standard error of the mean (SEM) for N≧3. Statistical analysis was performed using one-way ANOVA + Dunnett's multiple comparison, two-way ANOVA + Sidak's multiple comparison, or an independent t-test using an assumed Gaussian distribution. P≦0.05 was considered statistical significance. * =p≦0.05, ** =p≦0.01, *** =p≦0.001, ****(=p ≤ 0.0001). All statistical analyses and models were performed using GraphPad Prism 9.

[0116] result Production and sequencing of stable PDE4D7 knockdown LNCaP Stable PDE4D7 knockdown LNCaP cell lines were produced by shRNA-mediated lentiviral transduction. Several knockdown clones were analyzed for PDE4D7 expression, and qPCR revealed that P1 cells exhibited the most significant PDE4D7 mRNA-mediated downregulation (Figure 7). P1 cells, along with WT and scrambled-shRNA LNCaP cell lines, were selected to investigate the effects of PDE4D7 expression in prostate cancer. Significant evidence of PDE4D7 knockdown at the mRNA [Figure 1A] and protein levels [Figure 1B] was obtained in P1 LNCaP cell lines compared to WT cells. The reduction in PDE4D7 protein expression was also confirmed using confocal microscopy [Figure 1C].

[0117] Changes in genomic profiles between WT, scrambled, and P1 cells were analyzed using NGS RNA-seq. Top-expressed genes [Figure 1D] were shown as z-scores between WT and P1 cells, with many of these genes exhibiting significant downregulation in P1 LNCaP. Advaita pathway analysis was performed on the RNA-seq data, identifying that top-expressed genes are involved in PCa-related signaling pathways [Figure 1E], and that logFC is most significantly downregulated for KLK3, AR, SPINT1, TMPRSS2, and NKX3.1. Western blotting was performed on selected proteins to confirm that the RNA-seq data reflected the protein level. Very significant downregulation of all selected proteins [Figure 1F] was observed in P1 cell lines compared to WT. Importantly, scrambled-shRNA cell lines did not show any notable differences in the expression of these proteins (Figure 8).

[0118] PDE4D7 knockdown in LNCaP cells alters the phenotype and confers resistance to enzalutamide. Knockdown of PDE4D7 in the LNCaP cell line P1 significantly enhanced the cell's proliferative characteristics. To confirm that the observed effect was due to PDE4D7 knockdown and not lentiviral incorporation, xCELLigence proliferation assays were also performed on WT, P1, and scrambled-shRNA knockdown (SC2) LNCaP cell lines. Real-time proliferation curves showed enhanced proliferation of P1 cells compared to both WT and SC2 [Figure 2A], which correlated with a significant difference in gradient analysis. Importantly, there was no significant difference in proliferative characteristics between WT and SC2 LNCaP cells.

[0119] Enzalutamide is an AR antagonist used to treat advanced PCa (Menon and Higano, 2013). Given that significant downregulation of PDE4D7 expression is characteristic of androgen-insensitive prostate cancer (Henderson et al., 2014), we wanted to investigate the effect of enzalutamide treatment on the proliferation of WT and PDE4D7-deactivated PCa cells. To determine the optimal therapeutic concentration in WT and P1 LNCaP cells, dose-response experiments were conducted by increasing the drug concentration and monitored using xCELLigence technology. All concentrations tested resulted in a significant decrease in WT LNCaP cell proliferation (0.1–30 μM), with concentrations above 1 μM resulting in minimal proliferation [Figure 2B]. In contrast, P1 LNCaP cells showed minimal reduction in cell proliferation as the dose of enzalutamide increased [Figure 2C], and even exhibited a significant enhancement of proliferation compared to DMSO control as the concentration increased to 30 μM. Overall, these data strongly suggest that PDE4D7 knockdown in LNCaP cells confers resistance to enzalutamide.

[0120] Given the significant difference in AR expression between WT and P1 cells [Figures 1E and 1F], we decided to test the expression of the most relevant AR variant (AR-V7), but the results did not reveal any clear difference in expression [Figure 2D]. RNA-seq analysis also identified CCND1 as a gene that was significantly downregulated in P1 cells compared to WT [Figure 1E].

[0121] Cyclin D1 regulates androgen-dependent transcription and cell cycle progression [REF]. Therefore, given that AR is downregulated in P1 cells, it is not surprising that CCND1 is also downregulated [Figure 2F]. Interestingly, however, cyclin B1 appears to be upregulated in P1 cells compared to WT cells [Figure 2E]. This may represent a compensatory mechanism in the cell involving upregulation of the expression of other cell cycle proteins to induce a clear enhancement of proliferation.

[0122] PDE4D7 knockdown in LNCaP cells confers resistance to PARP inhibition, and reintroduction of PDE4D7 leads to salvation. Olaparib is an FDA-approved poly(ADP-ribose) polymerase (PARP) inhibitor effective against LNCaP cells (Feiersinger et al., 2018). Ceraracertib is a telangiectatic ataxia mutation and Rad3-related (ATR) inhibitor (AZD6738), and has been suggested to enhance synergistic effects when combined with olaparib in mCRPC (Lloyd et al., 2020). In light of this, we wanted to determine whether PDE4D7 knockdown affects the LNCaP cell response to any of these treatments. Using real-time proliferation analysis, LNCaP WT proliferation was dose-dependently reduced by olaparib [Figure 3A], but the effect on PDE4D7-deficient LNCaP was minimal [Figure 3B]. Gradient analysis of growth rates showed a significant reduction in WT growth with 3, 10, and 30 μM olaparib, but similar data were obtained in cells with PDE4D7 knockdown, showing an unexpected reduction in the gradient at lower concentrations (0.3 and 1 μM). However, it is noteworthy that cells with reduced PDE4D7 expression were resistant to 10 and 30 μM olaparib [Figure 3B].

[0123] PARP cleavage is required for late-stage apoptosis; therefore, analysis of the PARP / cPARP ratio can be used to determine the induction of apoptosis in cells [REF]. Here, there is a reduction in the PARP / cPARP ratio between WT and P1 cells [Figure 3C].

[0124] Given that PDE4D7 knockdown LNCaP cells exhibited enhanced proliferation compared to WT cells [Figure 2A] and became resistant to PARP inhibition with olaparib [Figure 3C], we wanted to determine whether the phenotype could be salvaged by reintroducing PDE4D7 into P1 cell lines. First, we evaluated the real-time proliferation of P1 and P1-PDE4D7+ / + cells, and reintroducing PDE4D7 into P1 cells resulted in a significant downward regulation of neoplastic growth [Figure 3D]. Furthermore, P1-PDE4D7+ / + cells were more sensitive to olaparib treatment [Figure 3E], and statistically significant downward regulation of proliferation was recorded compared to both olaparib-treated P1 cells and DMSO-treated P1-PDE4D7+ / + cells, as previously demonstrated in WT LNCaP cells. This data reaffirms that the role of PDE4D7 is to slow PCa cell proliferation and to promote sensitivity to clinically relevant PCa chemotherapeutic agents.

[0125] PDE4D7 knockdown in LNCaP cells does not affect the response to ATR inhibition, but may limit the docetaxel-treated response. Docetaxel is a chemotherapeutic drug that induces DNA damage, and we decided to use this to find out if there is a difference between the ability of WT and P1 cells to repair DNA and thus survive treatment. Compared to DMSO, docetaxel significantly reduced the proliferation of both WT and PDE4D7 knockdown LNCaP at all concentrations except 10 μM in P1 cells, which appeared to have no effect (Figures 4A and 4B, respectively). Given that 10 μM was effective in WT but not in P1, we investigated how PDE4D7 reintroduction into P1 cells affects this. Similar to olaparib [Figure 3D], P1-PDE4D7+ / + cells were more sensitive to docetaxel treatment than P1 cells [Figure 4C], suggesting that PDE4D7 deficiency enhances the ability to repair associated DNA damage and protects against the induction of cell death.

[0126] Interestingly, PDE4D7 knockdown conferred resistance to 10–30 μM olaparib in LNCaP cells, yet these cells were highly sensitive to the same concentration of seracertib. Growth curves for WT and PDE4D7 knockdown LNCaP treated with gradually increasing concentrations of seracertib showed similar trends in sensitivity to the compound [Figures 4D and 4E, respectively]. Analysis of the curve slope between 0–30 h post-treatment yielded similar results for both cell types. In both cases, the most significant growth inhibition occurred with 10–30 μM treatment. These results highlight that suppression of PDE4D7 expression confers resistance to olaparib-mediated PARP inhibition, but these cells are highly sensitive to seracertib-mediated ATR inhibition.

[0127] PDE4D7 knockdown in LNCaP cells affects the response to many current anti-cancer compounds. Many of the RNAseq genes identified as specifically expressed in WT and P1 cells are involved in antiandrogen resistance. Glucocorticoid receptor (GR) signaling can bypass AR inhibition and lead to enhanced proliferation in CRPC patients [REF]. In contrast to the most specific gene thought to be downregulated in P1 cells, GR expression was upregulated in P1, and this was confirmed by Western blotting as significant protein upregulation [Figure 5A]. Therefore, we decided to analyze the response to mifepristone, a GR antagonist. Real-time proliferation analysis revealed that proliferation in both WT and P1 cells was not reduced by 0.3–10 μM mifepristone treatment compared to DMSO [Figures 5B and 5C, respectively]. However, 30 μM mifepristone successfully inhibited WT LNCaP proliferation, but at the same concentration, it had the opposite effect on P1 LNCaP, resulting in a significant enhancement of proliferation. Analysis of the proliferation curves shows that for P1 LNCaP, initial proliferation was inhibited by 30 μM mifepristone [Figure 5C: pink line] compared to lower concentrations, but this effect eventually disappeared, indicating that the cells regained their proliferative capacity.

[0128] Screening of the Prestwick Library against DuCaP and DU145 PCa cell lines revealed that antiparasitic drugs are active against these cells [Ralf data - including?]. The repurposing of these drugs, such as mebendazole, shows potential in the treatment of advanced PCa in combination with docetaxel [REF]. Analysis of the effects of mebendazole on WT and PDE4D7 knockdown LNCaP showed significant differences between cell line responses. Interestingly, WT cells showed a dose-dependent significant downregulation of cell proliferation [Figure 5D], while the effect on P1 cells was far more amplified and significant, with immediate cell death initiation in the cells at both concentrations [Figure 5E]. This data suggests that PDE4D7 knockdown confers sensitivity to mebendazole compared to WT LNCaP.

[0129] cAMP kinetics and the effects of PDE4 activators in response to PDE4D7 knockdown We investigated cAMP dynamics in the intracellular domain of WT and P1 LNCaP using genetically encoded cAMP-fluorescence resonance energy transfer (FRET) probes. The CUTie cAMP FRET probe has been shown to have several advantages over conventional constructs [REF]. CUTie probes targeting the cell membrane (AKAP-79) or cytosol (CYTO-pDUAL) were obtained from the Zaccolo lab (REF). Treatment with rolipram induced a downregulation of the FRET ratio in the membrane compartment of WT LNCaP, which indicates an increase in cAMP concentration. The combined effect of forskolin (adenylate cyclase activator) and IBMX (non-specific PDE inhibitor) treatment, which normally yields a maximal response, did not have any further effect on these cells, highlighting that PDE4 inhibition is responsible for maximizing cAMP concentration in the membrane of WT LNCaP [Figure 6B]. P1 LNCaP cells did not show a significant difference in the FRET ratio upon rolipram treatment, but exhibited a slight decrease in the ratio and an increase in intracellular cAMP upon forskolin / IBMX addition [Figure 6D]. Given that PDE4D7 is known to be substantially membrane-located [Figure 1C][REF], inhibition by rolipram leads to upregulation of cAMP at this site in WT cells but not in P1 cells, suggesting that basal cAMP levels may already be elevated because PDE4D7 is already knocked down. PDE4D7 is also known to be present in the cytosol, but no significant difference was observed between WT and P1 LNCaP cells upon rolipram treatment at this site; however, forskolin / IBMX addition appeared to decrease the FRET ratio and increase cAMP [Figures 6A and 6C].

[0130] Metformin is a well-known drug used to treat type 2 diabetes, but its potential in treating a number of cancers has shown promising results [REF]. Regarding PCa, metformin has been shown to enhance overall survival / time to recurrence, but remains controversial due to a lack of evidence that it treats / reduces PCa tumors themselves (He et al., 2019). Given that metformin is known to reduce cAMP production (Miller et al., 2013), we decided to test its effects between WT and P1 LNCaP. Analysis of proliferation phenotypes shows that metformin successfully significantly reduced proliferation in WT cells at all concentrations [Figure 6E], while in P1 cells, significant inhibition was observed only at the maximum concentration tested [Figure 6F].

[0131] ML-R2 is an activator compound of the long form of PDE4 that allosterically binds to the enzyme to mimic PKA phosphorylation in the UCR1 domain [REF]. Since PDE4D7 knockdown leads to an enhancement of the proliferation phenotype in LNCaP, we decided to test whether this could be rescued using a PDE4 activator. Our results revealed that neither concentration had any effect on proliferation in WT LNCaP [Figure 6G], but the addition of ML-R2 caused inhibition of cell proliferation in P1 cells [Figure 6H]. PDE4D7 knockdown in LNCaP leads to an enhancement of cell proliferation, but activation of PDE4 in these cells has an inhibitory effect on proliferative capacity, which again highlights the importance of the PDE4 family, particularly PDE4D7, in driving PCa. [Examples]

[0132] We analyzed stable P1-PDE4D7+ / +LNCaP cell lines using cell indicators (N=3, Figure 9). Stable reintroduction resulted in PDE4D7 protein expression, as confirmed by Western blotting. Stable reintroduction of PDE4D7 into P1 LNCaP reduced real-time proliferation. Using cell indicators and gradient analysis, a statistically significant downregulation of proliferation was observed in P14D7+LNCaP. Reintroduction of PDE4D7 in P1-PDE4D7 LNCaP cells was confirmed to reverse the knockdown phenotype (a completion of N=3 was just needed to plot the graph).

[0133] The long-acting PDE4 morphogenetic activator ML-R2 was introduced into WT, P1, and P14D7 cells, and real-time proliferation of LNCaP (N=3) was evaluated (Figure 10). MLR2 had no effect on the proliferation of WT LNCaP, MLR2 attenuated the proliferation of P1 LNCaP, and stable reintroduction of PDE4D7 into P1 LNCaP rescued the phenotype of WT LNCaP that was MLR2-sensitive.

[0134] P1 and P1+4D7LNCaP cells were treated with olaparib (N=3, Figure 11). As a control, P1+4D7 cells were treated with DMSO. A+B) Reintroduction of PDE4D7 into P1 cells enhances sensitivity to olaparib, but reintroduction of PDE4D7 alone reduces proliferation to the same extent.

[0135] We investigated PARP / cPARP expression in WT and P1 LNCaP+ / - olaparib / docetaxel-treated cells (N=3, Figure 12). Treatment with olaparib / docetaxel increased PARP cleavage in WT LNCaP, which correlates with the induction of apoptosis (as shown by xCELLigence). There was no increase in the cleaved PARP ratio in P1 LNCaP, which correlates with these cells not undergoing apoptosis in response to the treatment (correlated with xCELLigence).

[0136] Having described the present invention in full, it will be understood by those skilled in the art that the present invention can be implemented within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without unnecessary experimental methods.

[0137] While the present invention has been described in relation to its particular embodiments, it is understood that further modifications are possible. This application is intended to cover any variations, uses, or adaptations of the present invention, which, as a whole, adhere to the principles of the invention and fall within the known or customary practices within the art to which the invention belongs, and which include departures from the disclosure applicable to the essential characteristics of the foregoing as shown in accordance with the scope of the appended claims.

[0138] All references cited herein, including articles or abstracts of academic journals, published or corresponding patent applications, patents, or any other references, are fully incorporated herein by reference, including all data, tables, figures, and text presented in the cited references. In addition, the entire content of references cited within the cited references is also fully incorporated herein by reference.

[0139] The steps of known methods, steps of prior art, and references to known or prior art methods do not in any way constitute an assertion that any aspect, description, or embodiment of the present invention is disclosed, taught, or proposed in the relevant art.

[0140] The foregoing descriptions of specific embodiments fully illustrate the overall nature of the Invention, which allows others to readily modify and / or adapt various applications of such specific embodiments without departing from the overall concept of the Invention, without requiring any unnecessary experimental methods, by applying in-articulate knowledge in the Art (including the contents of the references cited herein). Therefore, such adaptations and modifications are intended to fall within the meaning and scope of equivalent embodiments of the disclosed embodiments, based on the teachings and guidance presented herein. Technical terms or expressions used herein are for illustrative purposes only, not limiting purposes, and it should be understood that such terms or expressions are to be interpreted by those skilled in the art in conjunction with the teachings and guidance presented herein. [Examples]

[0141] method Patient cohort and tissue samples This study included a total of 367 patients managed at two prostate cancer centers in Germany. Of these, 363 patients had at least one adverse pathological feature (PSM: positive surgical margin; SVI: seminal vesicle invasion; EPE: extraprostatic extension; LNI: lymph node invasion; Gleason 4 components) and / or were classified as intermediate or high risk of disease progression after treatment according to the clinical risk metrics CAPRA-S (24) and / or the EAU-BCR risk model (25), while four patients showed elevated PSA postoperatively without any of the specified adverse features. All 367 patients experienced biochemical recurrence during the post-treatment follow-up period and were scheduled for salvage radiotherapy (SRT) to the pelvic floor with a total radiation dose of 50–72 Gy in multiple fractions. Of the 367 patients, 188 also received antiandrogen therapy at various points in time (before, during, or after SRT).

[0142] Two small biopsy punches (approximately 1x2 mm) representing the typical resected tumor area were collected from tumor-defining lesions in 367 patients who underwent surgery between 1994 and 2011. Regional institutional ethics committees approved the collection of patient tissue for clinical research.

[0143] Production of stable, PDE4D7-modified LNCaP prostate cancer cells. - Stable shRNA-mediated PDE4D7 knockdown LNCaP LNCaP clone FGC (ATCC® CRL1740®) was mixed with 10% (v / v) fetal bovine serum and 1% (v / v) L - The cells were cultured in RPMI1640 medium (Gibco®) supplemented with glutamine and 1% (v / v) penicillin / streptomycin, and stored in an incubator at 37°C with 5% CO2.

[0144] The lentiviral transfection system's transport plasmid was designed to stably express an shRNA hairpin targeting the first coding exon of the PDE4D7 transcript. As a control, the scrambled nucleotide sequence of the PDE4D7-targeting shRNA was cloned into the lentiviral transport plasmid. LNCaP cloned FGC cells were infected with lentivirus at MOI=10 in the presence of 10 μg / ml polybrene. The cells were maintained in puromycin-supplemented medium before selection of stably transduced cells and clonal proliferation (see Supplementary Materials for details). RNA extraction and qPCR validation of gene knockdown were performed as described below.

[0145] - Inducible PDE4D7 expression in PDE4D7 knockdown LNCaP P1 cells The PDE4D7 (GenBank ID AF536976) sequence was first subcloned into a lentiviral vector containing CMV (LVR-1001-pLV-CMV-PGK-Puro), and then subcloned into a Tet-On-inducible vector pLV-tet-on-3G-P2A-Puro. LNCaP-shRNA P1 cells were then incubated with 10% (v / v) fetal bovine serum and 1% (v / v) L Cells were maintained in RPMI1640 medium supplemented with glutamine and 1% (v / v) penicillin / streptomycin, and infected with Tet-On-inducible lentivirus with an MOI of 5-10 in the presence of 8 μg / ml polybrene. Cells were maintained in puromycin-supplemented medium before selection and clonal proliferation of stably transduced cells (see Supplementary Materials for details). Transduced cells with Tet-On-inducible lentivirus were tested for induction of PDE4D7 expression in the presence of 1 μg / ml doxycycline. PDE4D7 expression was estimated by RT-PCR.

[0146] molecular analysis For downstream molecular analysis, the following cell lines / clones were used: LNCaP FCG wild-type (WT); LNCaP (clone SC2) transfected with scrambled shRNA control; LNCaP (clones P1 and w5.2) transfected with shRNA targeting PDE4D7; and LNCaP clone P1 (clone P1-TET4D7) transfected with Tet-On inducible PDE4D7 vector.

[0147] RT-qPCR analysis - Patient biopsy: To account for potential tumor heterogeneity, two tissue punches from the RP cohort were combined before nucleic acid extraction. Potential differences in tumor cell types were addressed by normalizing PDE4D transcript expression against four reference genes. All molecular laboratory methods used, including RNA extraction, quantitative real-time PCR (RT-qPCR), oligonucleotide primers / probes, and statistical analysis, were as previously described (21).

[0148] Cell line: RNA was extracted from the LNCaP cell line using the RNeasy Mini kit (QIAGEN), and one-step qRT-PCR was performed using the Superscript™ III Platinum™ One-Step qRT-PCR Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Experiments were performed using the Step-One™ Real-Time PCR System (Thermo Fisher Scientific) with the following parameters: 1 cycle of 30 minutes at 50°C and 5 minutes at 95°C, followed by 45 cycles of 15 seconds at 95°C and 45 seconds at 60°C. Primers and probes were purchased from Integrated DNA Technologies at 400 nM primer / 200 nM probe and 1 ng / μl RNA per reaction (supplementary method). 2 -ΔΔCt The Ct value was quantified using a specific method, and statistical analysis was performed on the ΔΔCt value.

[0149] Calculation of clinical and genomic risk scores - Calculation of the clinical risk score CAPRA-S The postoperative CAPRA-S risk score and its corresponding low (1), intermediate (2), and high (3) risk categories were calculated as previously described (24). The EAU-BCR score and its two categories of low and high postoperative risk for disease progression were determined as published (25).

[0150] - Calculation of PDE4D7 score and PDE4D7 score category Normalized PDE4D7 transcript expression was produced by subtracting the RT-qPCR Cq of PDE4D7 transcript from the mean RT-qPCR Cq of a reference gene, and this was converted into a PDE4D7 score and its associated PDE4D7 score class (21). In multivariate Cox and logistic regression analyses for various available biological and treatment-related outcomes, the PDE4D7 score was used as a continuous variable. In Kaplan-Meier survival analysis, the normalized PDE4D7 transcript expression values ​​were converted to a percentile distribution (pPDE4D7 score), and since all scores were distributed between 0 and 1, this allowed for more efficient comparison with other genomic scores (see also below). Two cutoffs for the pPDE4D7 score were defined by AUROC (Area Under the ROC Curve) analysis, using prostate cancer-specific mortality during the 5 years after the start of SRT as the dependent variable and the pPDE4D7 score as the independent variable. One cutoff (pPDE4D7>0.2) was defined as the point in AUROC with maximum sensitivity and specificity. The other cutoff was defined as maximum sensitivity (pPDE4D7>0.87). As a result, the pPDE4D7 score classified stratified patients into three subcohorts: pPDE4D7 score>0.87: "high"; pPDE4D7 score>0.2 and <=0.87: "moderate", and pPDE4D7 score <=0.2: "low".

[0151] - Calculation of cell cycle progression and genomic prostate score To derive risk scores for two commercially available prognostic studies using 31 gene signatures for CCP (Cell Cycle Progression) and 12 gene signatures for GPS (Genome Prostate Score), mxCCP and mxGPS scores were inferred from RNA-seq expression data of each target and reference gene based on a previously published formula (26) for calculating risk scores and following a previously published protocol. The derived scores were converted to percentile distributions (mxpCCP and mxpGPS, respectively). Where indicated, either mxCCP and mxGPS (AUROC analysis) or mxpCCP and mxpGPS (Cox regression and Kaplan-Meier survival) were used for use in downstream statistical analysis.

[0152] RNA sequencing was performed as described in Example 1.

[0153] Gene Set Enrichment Analysis (GSEA) Gene set enrichment analysis (GSEA) was performed as described here (https: / / www.gsea-msigdb.org / gsea / index.jsp). A gene expression characteristic count matrix derived from RNA-seq was used as input for the enrichment analysis.

[0154] Whole-genome sequencing (WGS) Approximately 100 ng of gDNA per sample was randomly fragmented at 350 bp points by sonication. Libraries were constructed according to the TruSeq DNA Nano protocol (Illumina). In short, the sheared gDNA was end-prepped, dA-terminated, and fragments of approximately 350 bp were enriched using AMPure XP beads (Beckman Coulter) for size selection. A total of 8 cycles of indexing PCR were performed on the size-selected fragments, and the products were purified using AMPure XP beads. The quality of the final libraries was investigated using a LabChip GX nucleic acid analyzer (PerkinElmer). The concentration was determined by qPCR on the samples using 2X KAPA HiFi HotStart ReadyMix (Kapa Biosystems) as the master mix and a diluted PhiX sequencing control v3 as the standard. High-coverage sequencing was performed using an Illumina Novaseq 6000 system with an S4 flow cell and PE150 format.

[0155] Plasmid DNA transfection, xCELLigence real-time cell analysis, Western blotting, and immunofluorescence were performed as described in Example 1.

[0156] Data analysis and statistics Multivariate Cox regression and Kaplan-Meier analysis were applied to correlate PDE4D7 scores or clinical metrics (CAPRA-S score, EAU-BCR risk score) and their categories with prostate cancer or all-cause mortality in the total patient cohort (n=367). Statistical analysis was performed using the MedCalc software package (MedCalc software v2.20 BVBA, Ostend, Belgium), using the statistical tests indicated in the main text and / or figure captions of the manuscript. For LNCaP data, values ​​are presented as mean ± mean standard error (SEM) or standard deviation (SD) for N≧3 unless otherwise specified. Statistical analysis was performed using one-way ANOVA + Dunnett's multiple comparison, two-way ANOVA + Sidak's multiple comparison, or an unpaired t-test using an assumed Gaussian distribution. P≦0.05 was considered statistical significance. * =p≦0.05, ** =p≦0.01, *** =p≦0.001, **** (=p ≤ 0.0001). All statistical analyses and models were performed using GraphPad Prism 9.

[0157] result The PDE4D7 score is associated with survival outcomes in high-risk prostate cancer patients. All participants enrolled in this study had experienced postoperative PSA recurrence and were subsequently stratified based on salvage radiotherapy (SRT). Pathological analysis of surgical specimens revealed pT3a or higher-order disease in nearly 80% of the 367 patients (Table 3). Extraprostatic extension and positive surgical resection margins were identified in 62.4% and 72.2% of cases, respectively. Based on the CAPRA-S risk score, the majority of patients (85%) were classified as intermediate-risk (41.1%) or high-risk (44.1%) for postoperative disease progression. More than half of the patients (188 / 367) received androgen deprivation therapy (ADT) at some point during the study period. During a median follow-up period of 103 months after prostatectomy, 11.7% of participants died from prostate cancer and 17.4% died from any cause (Table 3).

[0158] Multivariate Cox regression analysis was performed to determine the association between PDE4D7 score and prostate cancer-specific mortality (PCSM) after initiation of SRT, adjusting for pathological prognostic variables (i.e., ISUP Gleason grade group, pathological pT stage, extraprostatic extension and surgical margin status, seminal vesicle involvement, and lymph node invasion). A strong inverse correlation was observed between PDE4D7 score and PCSM after initiation of SRT in a multivariate model including clinical prognostic parameters, as previously reported for biochemical recurrence as a clinical endpoint (HR=0.37; 95% CI 0.23~0.58; log-rank p<0.0001) (Table 2). To determine whether the PDE4D7 score provided independent prognostic information in the context of genomic mxpCCP or mxpGPS signature, these scores were included separately in the multivariate analysis. Interestingly, the mxpCCP score was not significantly associated with PCSM in this cohort (p=0.17), which may be due to limitations in inferring risk scores from RNA-seq data (see Methods). The mxpGPS signature showed a borderline significant association with PCSM (HR=4.0; 95% CI 0.93–17.5; log-rank p=0.06). In both analyses, the PDE4D7 score remained a significant variable in the multivariate models (p=0.003 and p=0.002). The PDE4D7 score was also tested in combination with the clinical prognosis models CAPRA-S and EAU-BCR risk scores in multivariate analyses for PCSM. In both models, the PDE4D7 score remained the most significant predictor of PCSM after SRT (HR=0.36, p<0.0001; HR=0.31, p<0.0001, respectively).

[0159] Kaplan-Meier (KM) analysis of pPDE4D7 percentile scores stratified patients into three subcohorts based on statistically significant survival rates (log-rank p<0.0001; Figure 13A). Patients in the high pPDE4D7 score class had a 100% survival rate after SRT, while patients in the intermediate and low score classes had survival rates of approximately 90% and less than 50% 10 years after SRT, respectively (Figure 1A). All-cause mortality showed a similar trend in this patient cohort (Figure 13B). The EAU-BCR risk model stratified patients into low and high-risk groups, with the low-risk group having significantly better survival rates at 10 years post-radiation (HR=3.7; 95% CI 2.0~6.8; log-rank p<0.0001; Figure 13C). The survival rate in the low-risk group was over 90%, while in the high-risk group it was approximately 72%. The CAPRA-S model did not show significant stratification between risk groups.

[0160] To assess the predictive ability of the PDE4D7 score for 5-year PCSM after salvage radiotherapy, receiver operating characteristic (ROC) curve analysis was performed, and the corresponding area under the curve (AUC) was calculated. The PDE4D7 score was compared to individual clinical parameters and clinical models, as well as to a combined model incorporating the PDE4D7 score along with the EAU-BCR risk score and further clinical variables (ISUP p-Gleason grade group, p-T stage, extraprostatic extension and surgical margin status, SVI, and LNI). Individual clinical parameters and clinical models showed an AUC of less than 0.7, while the PDE4D7_clinical combined model showed an AUC of 0.81 (PDE4D7_EAU-BCR) and 0.87 (full risk model) (p<0.0001). These results suggest that the PDE4D7_clinical combined model has superior predictive ability for 5-year PCSM compared to individual clinical parameters and models.

[0161] Finally, we applied Kaplan-Meier (KM) survival analysis to investigate whether the pPDE4D7 percentile score class could stratify patients who received antiandrogens in addition to SRT into distinct subgroups with varying risks of PCSM or ACM. Our results revealed statistically significant differences in survival outcomes between pPDE4D7 score classes (log-rank p=0.0004; Figure 13D). In particular, patients in lower pPDE4D7 score classes had a median survival of 89.6 months and an overall cancer-specific survival rate of 30% after initiation of androgen deprivation therapy (ADT), while patients in higher pPDE4D7 score classes did not reach a median survival. A similar pattern was observed for ACM, with overall survival declining to below 20% in the lowest pPDE4D7 class (Figure 13E). In contrast, none of the other risk classification models studied (mxpGPS, EAU-BCR risk, CAPRA-S) demonstrated significant performance in this setting, failing to stratify patients into various risk classes.

[0162] Our data show that the PDE4D7 score is a significant predictor of disease-specific (PCSM) and overall (ACM) mortality in patients with recurrent and advanced prostate cancer after primary surgical resection. Patients with higher PDE4D7 scores had more favorable long-term survival rates, while those with lower scores had less favorable outcomes regarding postoperative treatment survival. These findings suggest that the PDE4D7 score may be a useful predictor of patient outcomes in this patient population.

[0163] PDE4D7 knockdown is associated with loss of androgen signaling, enrichment of mesenchymal epithelial transition, and neuroendocrine prostate cancer.

[0164] To further investigate the biological significance of PDE4D7 in prostate cancer development and progression, we produced derivatives of the LNCaP (clone FCG) prostate cancer cell line by selective knockdown of PDE4D7 expression using a lentiviral-mediated shRNA strategy. Among several PDE4D-knockdown LNCaP clones, LNCaP_P1 cells showed the most significant downregulation of PDE4D7 mRNA expression, as confirmed by RT-qPCR. Western blot analysis and confocal microscopy further confirmed the reduction in PDE4D7 protein expression in LNCaP_P1 cells compared to wild-type LNCaP and scrambled shRNA control (LCNaP_SC2) (Figures 1A, 1B, and 1C). These findings demonstrate the success of producing a PDE4D7 knockdown model in LNCaP cells.

[0165] Next-generation sequencing (NGS) RNA sequencing was performed on two control cell lines (LNCaP_WT and LNCaP_SC2), as well as three PDE4D7 knockdown clones LNCaP_P1, LNCaP_w5.2, and LNCaP_w6.3. Corresponding gene expression count tables were used as input for gene set enrichment analysis (GSEA) of the 50 GSEA Hallmark pathway (gsea-msigdb.org). For pathway analysis in phenotypic wild-type (WT) and shRNA scrambled control SC2 versus PDE4D7 knockdown cell lines, the androgen response (AR) pathway was identified as the most significantly enriched gene set (false discovery rate (FDR) q value < 0.001; nominal enrichment score -2.1; Figure 1D, Figure 1E). To confirm that RNA-seq data reflected at the protein level, Western blotting was performed on selected PCa-related proteins, and significant downregulation was observed in the P1 cell line compared to the WT (Figure 1F). Importantly, the scrambled shRNA cell line SC2 did not show any notable differences in the expression of these genes. Among the genes most downregulated in the two PDE4D7 knockdown cell lines were the known AR-regulating kallikrein-related peptidases KLK2 and KLK3, transmembrane serine protease 2 (TMPRSS2), and the transcription factor NK3 homeobox-1 (NKX3-1). Furthermore, AR itself—though not part of this Hallmark AR response pathway—was also strongly downregulated in the PDE4D7 knockdown cell line P1 (Figure 1F). The findings described suggest that selective removal of PDE4D7 transcript expression leads to a cellular phenotype corresponding to androgen-insensitive proliferation.

[0166] In contrast, we identified several Hallmark pathways that were significantly enriched in PDE4D7 knockdown LNCaP derivatives. Of these, the EMT pathway showed the highest level of enrichment (FDR q value < 0.001; nominal enrichment score 1.9; Figure 14, Figure 1H). Other Hallmark pathways with FDR q value < 0.1 that may further contribute to the progressive phenotypes of P1 and w5.2 and are potentially relevant in clinical practice are the WNT beta-catenin and Hedgehog signaling pathways. Both pathways have previously been associated with the progression of prostate cancer after the disease has progressed to hormone resistance.

[0167] NED in prostate cancer is a hallmark of high-grade, AR-independent, treatment-resistant disease. It occurs in both primary and metastatic prostate cancer. NED is characterized by decreased AR expression and increased expression of neuroendocrine markers.

[0168] Given the importance of NED in high-grade prostate cancer, we evaluated the range of genes whose expression was altered in NEPC in our LNCaP model. This analysis revealed that many genes with NEPC-induced expression are upregulated in PDE4D7 knockdown models containing classical markers of NED, such as ENO2, SYP, AURKA, or NCAM1 (Figure 1I).

[0169] To explore the potential clinical relevance of these findings, we also analyzed the expression of key markers for Hallmark AR-responsive genes and NED in RNA-seq data from 533 human patient samples previously described by us. We found that all tested AR-responsive genes were downregulated along with reduced PDE4D7 expression in these patient tumors, but AR itself did not alter their expression. Similarly, we found that several markers reported in NED altered expression in low-to-high PDE4D7 patient tumors, and that the modulation followed those previously reported in NEPC.

[0170] PDE4D7 knockdown in LNCaP cells alters the phenotype and confers resistance to AR antagonists. Knockdown of PDE4D7 in the LNCaP cell line P1 resulted in increased cell proliferation characteristics, as confirmed by a real-time proliferation assay comparing P1 PDE4D7 knockdown cells to WT and SC2 LNCaP cells (Figure 2A). Importantly, there was no significant difference in proliferation characteristics between WT and SC2 LNCaP cells.

[0171] We investigated the effect of enzalutamide, an AR antagonist used to treat advanced PCa, on the proliferation of PCa cells with deactivated PDE4D7 expression. Dose-response experiments using real-time proliferation technology showed a significant reduction in WT LNCaP cell proliferation at all concentrations tested (0.1–30 μM), with the most significant effect at concentrations above 1 μM (Figure 2B). In contrast, P1 LNCaP cells showed minimal reduction in proliferation upon enzalutamide treatment, and even increased proliferation at higher concentrations (Figure 2C). Re-expression of PDE4D7 in LNCaP P1 cells using a Tet-On-inducible vector rescued the proliferation phenotype and restored sensitivity to enzalutamide (Figures 15A, 15B). These data strongly suggest that low PDE4D7 expression in PCa cells confers resistance to anti-androgen drugs such as enzalutamide.

[0172] To mimic PKA phosphorylation in the UCR1 domain, MR-L2, a long-form activator of PDE4 that allosterically binds to the PDE4D enzyme, was tested to rescue the enhancement of the proliferation phenotype induced by PDE4D7 knockdown in LNCaP. The results showed inhibition of proliferation in P1 cells but no effect on proliferation in WT LNCaP (Figure 2E, Figure 2F). This data suggests that activation of PDE4 in PDE4D7 knockdown LNCaP has an inhibitory effect on the proliferative capacity of LNCaP, and further highlights the importance of PDE4D7 in driving PCa.

[0173] PDE4D7-specific knockdown in LNCaP is associated with altered expression of DNA damage repair genes. Multiple studies have conclusively demonstrated that genomic alterations in the DDR gene are prevalent in both primary and metastatic prostate cancer tissue, leading to the clinical use of PARP inhibitors (PARPi) in patients with DDR deficiency. However, the emergence of resistance mechanisms has been observed in patients treated with PARPi. To investigate the potential effects of PDE4D7 knockdown on DNA repair mechanisms, we utilized the REACTOME homology-directed repair of a set of DNA double-strand break genes (reactome.org:R-HSA-5693538) and extended it with additional genes from other DNA repair pathways previously reported to be altered in prostate cancer. Our analysis of the expression of these genes across LNCaP wild-type and SC2 cell lines, as well as their PDE4D7 knockdown derivatives, revealed alterations in the expression of HDR genes in knockdown cells, particularly in subsets of DDR genes that are typically mutated in prostate cancer. Most of these genes were significantly enhanced in expression in PDE4D7 knockdown cells, but no significant differences were identified in single-nucleotide variants between parental and knockdown cell lines.

[0174] To further evaluate the correlation between low PDE4D7 expression and potential resistance to PARPi treatment, we re-analyzed previously obtained RNA-seq data from 533 human clinical samples, focusing on the expression of prostate cancer-related DDR genes. Our analysis revealed the most significant increase (p<0.001) in BRCA2 expression (BRAC2) accompanied by decreased PDE4D7 expression in patient tumors, along with a range of other DDR-related genes (Figure 16). BRCA1, ATM, and BRIP1 were also upregulated, but not to a significant level (not shown). Based on these findings, we hypothesize that altered DDR gene expression accompanied by decreased PDE4D7 transcription contributes to resistance to PARPi-mediated DDR targeting, and that cell lines with low PDE4D7 expression after selective knockdown may exhibit resistance to PARPi compounds such as olaparib.

[0175] PDE4D7 knockdown in LNCaP confers resistance to PARP inhibition and is rescued upon reintroduction of PDE4D7. Olaparib is an FDA-approved PARPi effective against LNCaP cells. Ceraracertib is a Rad3-related (ATR) inhibitor (AZD6738) for ataxia telangiectasia mutations, and has been proposed to enhance synergistic effects in combination with olaparib in metastatic CRPC. In light of this, we wanted to determine whether PDE4D7 knockdown affects the LNCaP cell response to either of these treatments. Using real-time proliferation analysis, LNCaP WT proliferation was dose-dependently reduced by olaparib (Figure 3A), but the effect on PDE4D7-deficient LNCaP was minimal (Figure 3B). Gradient analysis of proliferation rates showed significant reductions in WT proliferation with 3, 10, and 30 μM olaparib, but similar data were obtained in PDE4D7-knockdown cells, showing an unexpected reduction in the gradient at lower concentrations (0.3 and 1 μM). However, it is noteworthy that cells with reduced PDE4D7 expression were resistant to 10 and 30 μM olaparib (Figure 3B). Next, we attempted to determine whether the phenotype could be rescued by transient reintroduction of PDE4D7 into P1 cell lines. Similar to the TET-On induction of PDE4D7 in P1 cell lines, transient transfected P1-PDE4D7 high The cells revealed significant downward regulation of proliferation (Figure 3D). Furthermore, P1-PDE4D7 high The cells became sensitive again to 10 μM olaparib treatment (Figure 3E), and as previously demonstrated in WT LNCaP cells, olaparib-treated P1 cells and DMSO-treated P1-PDE4D7 cells were also sensitive. + / + A statistically significant downward regulation of cell proliferation was recorded compared to both groups. This data reaffirms that the role of PDE4D7 is to slow PCa cell proliferation and promote sensitivity to clinically relevant PCa therapeutics.

[0176] PDE4D7 knockdown in LNCaP cells does not affect the response to ATR inhibition, but may limit the docetaxel-treated response. Docetaxel is a DNA-damaging cytotoxic drug clinically used in the treatment of advanced prostate cancer. Compared to DMSO, docetaxel significantly reduced the proliferation of both WT and PDE4D7 knockdown LNCaP cells at all concentrations except 10 μM in P1 cells, where it appeared unresponsive. Given that 10 μM was effective in WT cells but not in P1 cells, we investigated the effect of PDE4D7 reintroduction into P1 cells on this. Similar to olaparib, P1-PDE4D7 + / + The cells were more sensitive to docetaxel treatment than P1 cells, suggesting that PDE4D7 deficiency enhances the ability to repair associated DNA damage and protects against the induction of cell death. This is consistent with the increased expression of several key DNA repair genes in PDE4D7 knockdown LNCaP cells.

[0177] Interestingly, PDE4D7 knockdown conferred resistance to 10–30 μM olaparib in LNCaP cells, yet these cells were highly sensitive to ATR inhibition by the same concentration of seracertib. Growth curves for WT and PDE4D7 knockdown LNCaP cells treated with gradually increasing concentrations of seracertib showed similar trends in sensitivity to the compound (Figures 17A and 17B, respectively). In both cases, the most significant growth inhibition occurred with 10–30 μM treatment. These results highlight that suppressing PDE4D7 expression confers resistance to olaparib-mediated PARP inhibition, but these cells remain highly sensitive to seracertib-mediated ATR inhibition.

[0178] Given that both ceraracertib and metformin appear to inhibit LNCaP cell proliferation regardless of PDE4D7 expression status (wt vs. P1, see Examples 1 and 3), ceraracertib or metformin may offer an alternative treatment option for prostate cancer patients resistant to antiandrogens (e.g., enzalutamide) or PARP inhibitors (e.g., olaparib) when the tumor has low PDE4D7 expression.

[0179] [Table 3] JPEG2026513644000008.jpg255124JPEG2026513644000009.jpg72121

[0180] [Table 4]

Claims

1. Products for use in the treatment, prevention, or remission of prostate cancer by reducing therapy resistance in subjects requiring treatment, prevention, or remission, wherein the products induce the expression of PDE4D7 or promote the activity of phosphodiesterase 4D7, and are selected from the following: A polynucleotide encoding a peptide having the sequence of SEQ ID NO: 2, or a variant thereof, wherein the variant is a polynucleotide encoding a peptide having a sequence having at least 90% sequence homology to SEQ ID NO: 2, and the peptide has phosphodiesterase activity, or A polynucleotide comprising the nucleotide sequence defined in SEQ ID NO: 1, or a variant thereof, wherein the variant comprises a nucleotide sequence having at least 90% sequence homology to SEQ ID NO: 1, and the polynucleotide is a variant encoding a peptide having phosphodiesterase activity, or A peptide comprising the peptide sequence defined in SEQ ID NO: 2, or a variant thereof, wherein the variant comprises a peptide sequence having at least 90% sequence homology to SEQ ID NO: 2, and the peptide has phosphodiesterase activity, or A peptide comprising a peptide sequence encoded by a polynucleotide comprising the nucleotide sequence defined in SEQ ID NO: 1, or a variant thereof, wherein the variant comprises a peptide sequence encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence homology to SEQ ID NO: 1, and the polynucleotide encodes a peptide having phosphodiesterase activity, or A compound defined by formula I, 【Chemistry 4】 During the ceremony, R １ is H, (C1-4) alkyl or (C1-4) alkyloxy, R ２ and R ６ It is selected independently of H, R ３ 、 R ４ and R ５ are independently selected from H, halogen, CN, (C １～４ )alkyl and (C １～４ )alkyloxy, and the (C １～４ )alkyl and (C １～４ )alkyloxy groups may be substituted with 1 to 3 fluoros. R ７ , R ８ , and R １０ It is selected independently from H and F, R ９ The compound is selected from (C1-4) alkyl, (C1-4) alkyloxy, CN, and halogen, and the (C1-4) alkyl and (C1-4) alkyloxy groups may be substituted with 1 to 3 fluorocarbons. or A peptide having a sequence containing a sequence selected from SEQ ID NOs: 24, 25, 26, or 27, or a sequence consisting of a sequence selected from SEQ ID NOs: 24, 25, 26, or 27, or a polynucleotide encoding a peptide having a sequence containing a sequence selected from SEQ ID NOs: 24, 25, 26, or 27, or a sequence consisting of a sequence selected from SEQ ID NOs: 24, 25, 26, or 27.

2. The aforementioned therapy resistance includes resistance to antiandrogen drugs, resistance to PARP inhibitors, and / or resistance to chemotherapeutic agents. Preferably, the antiandrogen drug resistance is resistance to enzalutamide, flutamide, nilutamide, bicalutamide, topirutamide, apalutamide, darolutamide, cimetidine, ketoconazole, proxalutamide (GT-0918), ceviteronel (VT-464), or abiraterone, more preferably resistance to enzalutamide, and / or Preferably, the PARP inhibitor resistance is resistance to olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib (BGB-290), rucaparib, CEP9722, or E7016, and more preferably, the therapy resistance is resistance to olaparib, and / or Preferably, the resistance to the chemotherapeutic agent is resistance to a chemotherapeutic agent selected from paclitaxel, docetaxel, Abraxane, Taxotere, cabazitaxel, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, barrubicin, estramustine phosphate, allestramustine, atrimustine, cistestrol acetate, estradiol mustard, estramustine, estromustine, ICI-85966, or fenestrol, and more preferably, the resistance to the therapy is resistance to osetaxel, cabazitaxel, mitoxantrone, or estramustine. A product for use according to claim 1.

3. The product for use according to claim 1 or 2, wherein the therapy-resistant prostate cancer has reduced expression of PDE4D7.

4. The product for use according to any one of claims 1 to 3, wherein the product is delivered using nanoparticles.

5. The product for use according to any one of claims 1 to 4, wherein the therapy resistance is resistance to antiandrogen drugs, and the treatment, prevention or remission further comprises administering an antiandrogen drug, preferably selected from enzalutamide, flutamide, nilutamide, bicalutamide, topirutamide, apalutamide, darolutamide, cimetidine, ketoconazole, proxalutamide (GT-0918), ceviteronel (VT-464), or abiraterone.

6. The product for use according to any one of claims 1 to 5, wherein the therapy resistance is PARP inhibitor resistance, and the treatment, prevention or remission further comprises administering to the subject a PARP inhibitor, preferably selected from olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib (BGB-290), rucaparib, CEP9722, or E7016.

7. The product for use according to any one of claims 1 to 6, wherein the therapeutic resistance is resistance to a chemotherapeutic agent, and the treatment, prevention or remission further comprises administering to the subject a chemotherapeutic agent, preferably selected from paclitaxel, docetaxel, abraxane, taxotere, cabazitaxel, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, barrubicin, estramustine phosphate, allestramustine, atrimustine, cistol acetate, estradiol mustard, estramustine, estromustine, ICI-85966, or fenestrol.

8. The product for use according to any one of claims 1 to 7, wherein the product is administered to a subject when the subject exhibits therapy resistance or low expression of PDE4D7 in a tumor.

9. The treatment, prevention or remission comprises preparing a sample derived from the subject and determining the expression level of PDE4D7, wherein the product is preferably administered to the subject when a low expression level of PDE4D7 is observed in the sample, according to any one of claims 1 to 8.

10. A compound for use in the treatment, prevention, or remission of prostate cancer in subjects requiring treatment, prevention, or remission, wherein the compound is an ATR inhibitor or a metabolic inhibitor, and the use is When high expression of PDE4D7 is observed in tumors, Low expression of PDE4D7 is observed, but PDE4D7 activity does not increase sufficiently to make the tumor sensitive again to inhibitor treatment, or In the case of metastatic tumors A compound for use, comprising administering the aforementioned compound to a person.

11. The ATR inhibitor is selected from RP-3500, seracertib (AZD6738), AZ20, elimusertib (BAY-1895344), elimusertib (BAY-1895344) hydrochloride, SKLB-197, ETP-46464, belzocertib (VE-822), dactricib (BEZ235), VE-821, torin 2, or CGK733, and more preferably the ATR inhibitor is seracertib, the compound for use according to claim 10.

12. The metabolic inhibitor is selected from methotrexate, pemetrexed, 6-mercaptopurine, 6-thioguanine, capecitabine, 5-fluorouracil, gemcitabine, cytarabine, leflunomide, CB-839, PEG-BCT-100 (ADIPEG20), AEB-1102, L-asparaginase, TVB-2640, AG-120 (ivosidenib), IDH305, BAY1436032, FT-2102, AG-221 (enasidenib), AG-881, AZD3965, CPI-613, or metformin, and more preferably the metabolic inhibitor is metformin, the compound for use according to claim 10.

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