Compound capable of inhibiting trim24 activity for use in methods of treating prostate and breast cancer

Abstract

The present invention relates to compounds capable of inhibiting TRIM24 activity and their use in combination with androgen deprivation therapy and / or androgen receptor signaling inhibitors and / or any type of AR inhibitor (e.g., pharmacological AR degrader) in a method of treatment of a hormone-sensitive metastatic prostate cancer, in particular in prostate cancer that are characterized by recurrent point mutations in SPOP. Moreover, the invention posits the use of inhibiting TRIM24 activity for the treatment of AR-V7-driven castration-resistant prostate cancer (CRPC).

Description

[0001] COMPOUND CAPABLE OF INHIBITING TRIM24 ACTIVITY FOR USE IN METHODS OF TREATING PROSTATE AND BREAST CANCER

[0002] DESCRIPTION

[0003] Technical field of the invention

[0004] The present invention relates to compounds capable of inhibiting TRIM24 activity and their use in combination with androgen deprivation therapy and androgen receptor signaling inhibitors in a method of treatment of a hormone-sensitive metastatic prostate cancer, in particular in prostate cancer is characterized by recurrent point mutations in SPOP. In addition, the invention relates to compounds capable of inhibiting TRIM24 activity in prostate cancer resistant to ADT / ARSi and most notably expressing androgen receptor isoforms lacking the ligandbinding domain, such as but not restricted to AR-V7.

[0005] State of art

[0006] In prostate cancer, androgens bind to the androgen receptor (AR) which in turn activates an oncogenic gene expression program. Because AR signaling represents a key lineage-specific oncogenic path in prostate cancer, androgen deprivation therapy (ADT) remains the mainstay for prostate cancer treatment in an early metastatic setting. Over the last years, second- generation anti-androgens (e.g., enzalutamide) or androgen synthesis inhibitors (e.g., abiraterone) have shown efficacy in castration-resistant prostate cancer (CRPC). The latter androgen receptor signaling inhibitors (ARSi) are currently combined upfront with ADT in early metastatic setting. Nevertheless, a substantial subset of patients relapses and rapidly progress under ADT / ARSi to develop fatal CRPC with limited therapeutic options left. Thus, there is a clinical need for more efficient combination therapies in early hormone-sensitive metastatic disease and CRPC.

[0007] Summary of the Invention

[0008] The inventors surprisingly found that in an ADT / ARSi-resistant xenograft model of CRPC(LNCaP-95) TRIM24 degradation inhibits tumor growth when the ARSi enzalutamide has no effect any- more, implying that TRIM24 degradation (dTRIM2430) may still have an effect when standard therapies targeting AR (ADT / ARSi) do not work anymore. At the molecular level, they show that dTRIM24 can inhibit AR signaling in CRPC. Most notably, the inventors consistently observe the inhibition of CRPC models driven by AR-V7 in the LNCaP-95 and 22Rvl cell lines. Mechanistically, the degradation of TRIM24 decreases both AR-V7 mRNA and protein and established target genes of AR- V7. levels at both the protein and mRNA levels. A similar effect is observed following the knockdown of TRIM24 in both cell lines (Fig. 1). Importantly, the inventor surprisingly showed a synergy between ADT / ARSi and TRIM24 knockdown or degradation in that the knockdown of TRIM24 or its degradation by dTRIM24 significantly downregulated AR signaling as evidenced by NKX3-1 in cells either exposed to ADT alone in charcoal -stripped serum or with the addition of the ARSi enzalutamide (ADT + ARSi) while not having any effect under androgen-rich conditions with dihydrotestosterone (DHT) (Fig. 2). Notably, dTRIM24 had similar effects in the AR-dependent cell lines LAPC4 and VCAP, highlighting its broad therapeutic potential.

[0009] In addition, the inventors found that also in vivo the parental castration-sensitive LNCaP xenograft model dTRIM24 synergizes with ADT (surgical castration) in delaying tumor relapse with higher efficacy than the ARSi enzalutamide. More efficient knockdown of TRIM24 using RNA interference (as compared to dTRIM24) completely abrogates tumor regrowth in this setting, suggesting that improved TRIM24 degraders may enhance the synergism between ADT / ARSi and TRIM24 degradation. At the molecular level, tumor regrowth is mediated by the re-activation of AR signaling and engagement of STAT3 signaling and EMT triggered by ADT. Thus, dTRIM24 or knockdown of TRIM24 inhibits both AR signaling and counteracts signaling pathways (e.g. STAT3 and EMT) that mediate resistance to ADT / ARSi (Fig. 3).

[0010] Moreover, the inventors show that ADT / ARSi treatment in cell culture trigger TRIM24- dependent activation of STAT3 phosphorylation and induction of STAT3 -related cytokine mRNA, which are being reverted by either degradation of TRIM24 or knockdown of TRIM24, respectively (Fig. 4). Key aspects of the results have been recapitulated also in VCaP and LAPC-4 cells (Fig. 4).

[0011] The inventors also showed that in an SPOP-mutant patient-derived xenograft model (LuCaP- 147) dTRIM24 even more efficiently blocks tumor relapse. The effect is again paralleled at the molecular level by repression of AR signaling and EMT (Fig. 5). Main advantages: Thus far, no in vivo activity has been reported using dTRIM24 in any tumor type. In the inventor's hands, the cell culture activities in prostate cancer cell lines have been rather modest and did not suggest the described in vivo activity. The experimental results of the inventors indicate that (i) dTRIM24 remains an effective treatment in AR-driven CRPC, especially driven by AR-V7 when ADT / ARSi does not work anymore, (ii) that dTRIM24 synergizes with ADT / ARSi as it not only further inhibits AR signaling under the combination of ADT and ARSi, but also counteracts adaptive mechanisms to AR inhibition which promote resistance thereof (EMT, STAT3), and (iii) that the synergy may be particularly strong in SPOP mutant prostate cancer.

[0012] Furthermore, the use of TRIM24 degradation may be transferable to endocrine-sensitive and resistant breast cancer based on the similarities of sex steroid hormone-driven cancers and prior findings suggesting an oncogenic role for TRIM24 in breast cancer.

[0013] Accordingly, the first object of the present invention is a compound capable of inhibiting TRIM24 activity for use in combination with androgen deprivation therapy and / or androgen receptor signaling inhibitors or any other type of AR inhibition (e.g., pharmacological AR degradation) in a method of treatment of a hormone-sensitive metastatic prostate cancer.

[0014] A further object of the present invention is a compound capable of inhibiting TRIM24 activity for use in combination with androgen deprivation therapy and / or androgen receptor signaling inhibitors or any other type of AR inhibition (e.g., pharmacological AR degradation) in a method of preventing or delaying tumor relapse and progression to castration-resistant disease.

[0015] A further object of the present invention is a compound capable of inhibiting TRIM24 activity for use in a method of treatment of castration-resistant prostate cancer (CRPC).

[0016] A further object of the present invention is a compound capable of inhibiting TRIM24 activity for use in a method of treatment of AR-V7-driven castration-resistant prostate cancer (CRPC) or other AR isoforms lacking the ligand binding domain.

[0017] A further object of the present invention is a compound capable of inhibiting TRIM24 activity for use in a method of treatment of a patient with castration-resistant prostate cancer, wherein the cancer of said patient is positive for the expression of AR-V7 protein or other AR isoforms lacking the ligand binding domain.

[0018] A further object of the present invention is a compound capable of inhibiting TRIM24 activity for use in a method of treatment of advanced endocrine-resistant breast cancer, in particular in combination with endocrine therapies, such for example the anti-estrogen tamoxifen.

[0019] In one preferred embodiment said prostate cancer is characterized by recurrent point mutations in SPOP.

[0020] A further object of the present invention is a pharmaceutical composition comprising a com- pound capable of inhibiting TRIM24 activity and a pharmaceutically acceptable carrier for any use herein disclosed.

[0021] Further features and advantages of the invention will appear from the following detailed description, which is provided for illustrative purposes only and not by way of limitation, with reference to the appended drawings.

[0022] Brief description of the drawings

[0023] Figure 1. 1A Immunoblot of TRIM24 and Tubulin in LNCaP-95 and 22RV1 cells following 48 hours of treatment with dTRIM24 at the indicated concentrations in cell culture. Treatment with DMSO (CNTR) was used as a reference. IB In the upper panel, immunoblot of TRIM24 and Tubulin in LNCaP-95 tumor following daily intraperitoneal application of dTRIM24 at lOmg / Kg for five days in vivo. The vehicle was used as a control (CNTR). In the lower panel, growth rate of LNCaP-95 xenografts in castrated NRG mice treated with vehicle (CNTR, n=6 mice), dTRIM24 (n=6 mice), and enzalutamide (ENZA) (n=6 mice). The treatment was given intraperitoneally daily, five times a week, for two weeks at 10 mg / kg for dTRIM24 and 30mg / kg for enzalutamide, respectively. The statistical significance of the results was tested using a two-way ANOVA test. 1C Weight of NRG mice bearing LNCaP-95 tumors during the treatment with vehicle (CNTR) and dTRIM24 (10 mg / kg) for two weeks. ID Enrichment scores of the indicated hallmark pathways using gene set enrichment analysis from RNA sequencing data of LNCaP-95 cells treated with DMSO (CNTR) or 5μM dTRIM24 for 48 hours in cell culture. The significance (loglO FDR) is shown by dot size. IE The knockdown of TRIM24 or its degradation decreased AR-V7 levels at both the protein and mRNA levels, downregulating AR and mTOR signaling. Immunoblot of the indicated proteins in LNCaP-95 and 22RV1 cells treated with 5 μM dTRIM24 for 3 days in charcoal-stripped serum (CSS) medium. Treatment with DMSO (control) was used as a reference. IF The dTRIM24 treatment led to a downregulation of the ARV7 target genes. Bar plot showing the qPCR analysis for these genes in LNCaP-95 and 22rvl cells over 3 days in CSS medium. Fold change is calculated against the control (DMSO), with mean values and standard error of the mean (SEM) from at least three independent experiments, underscoring the significance of our results via an unpaired Student’s t-test. 1G The degradation of TRIM24 reduces colony formation in ARV7-driven cell lines. A colony formation assay was conducted over 21 days for the LNCaP-95 and 22rvl cell lines treated with 5 μM dTRIM24 in CSS medium. The left panel presents a representative image of colony confluency, while the right panel illustrates the fold change compared to the control condition. This data is derived from four independent experiments, with the bars representing the mean and the Standard Error of the Mean (SEM). Statistical significance was assessed using an unpaired Student’s t-test. 1H-I The knockdown of TRIM24 in LNCaP-95 and 22Rvl cells has a similar effect to TRIM24 degradation. 1H A bar plot illustrates the qPCR analysis of ARV7 target genes in control (CNTR), TRIM24- KD1, and TRIM24-KD2 LNCaP-95 and 22RV1 cells over 3 days in CSS medium. The fold change is calculated relative to the control (DMSO), with mean values and standard error of the mean (SEM) derived from at least three independent experiments. The significance of our results was confirmed using a one-way ANOVA test. II a colony formation assay over 21 days for CNTR, TRIM24-KD1, and TRIM24-KD2 LNCaP-95 and 22RV1 cells treated with 5 μM dTRIM24. The left panel presents a representative image of colony confluency, while the right panel shows the fold change compared to the control condition. This data is based on four independent experiments, with bars indicating the mean and SEM. Statistical significance was assessed using a one-way ANOVA test.

[0024] Figure 2. 2A Immunoblot of TRIM24 and Tubulin in LNCaP, LAPC4, and VCAP cells following 48 hours of treatment with dTRIM24 at the indicated concentrations in cell culture. 2B-D The degradation of TRIM24 downregulated AR signaling and decreased colony formation under androgen deprivation conditions (ADT) following enzalutamide (ARSi) treatment in AR-dependent cell lines. 2B Immunoblot of TRIM24, AR, MYC, NKX3-1, and Tubulin in LNCaP, LAPC4, and VCAP cells maintained for 3 days in CSS with enzalutamide (CSS+ENZA) alone or in combination with dTRIM24 (CSS+ENZA+dTRIM24) at the indicated concentration. 2CD Colonies formation assay of LNCaP, LAPC4, and VCAP cells maintained for 21 days in CSS with 1 nM DHT (DHT), CSS medium alone (no DHT), and CSS medium with 10 μM ENZA (no DHT + ENZA), along with dTRIM24 treatments. The concentrations used for the dTRIM24 were 5 μM for LNCAP and LAPC4 cells and 1 μM for VCAP cells. In C is a representative image of colony confluency; in D, a bar plot shows fold change in colony confluency for both cell lines at the indicated condition. The fold change is calculated based on the average colony confluency observed in the DHT condition. This data is derived from four independent experiments, with the bars representing the mean and the Standard Error of the Mean (SEM). Statistical significance was evaluated using an unpaired Student’s t-test. 2E-F The knockdown of TRIM24 in LNCaP cells shows a similar effect of the TRIM24 degradation in ADT / ARSi condition. 2E Immunoblot analysis was conducted on the indicated proteins in LNCaP knockdown cells (TRIM24KD-1 and TRIM24KD-2) in comparison to control cells (shSCR and CNTR), maintained for 3 days in CSS medium with enzalutamide at the specified concentration. 2F Colony formation assay was performed on CNTR, TRIM24-KD1, and TRIM24-KD2 LNCaP cells, which were maintained for 21 days in CSS medium with 1 nM DHT, CSS medium alone (no DHT), and CSS medium with 10 μM enzalutamide (no DHT + ENZA). The left panel displays a representative image of colony confluency, while the right panel presents a bar plot illustrating the fold change in colony confluency.

[0025] Figure 3. 3 A Immunoblot of TRIM24 and Tubulin in LNCaP xenografts following daily intraperitoneal administration of dTRIM24 at lOmg / kg for five days. A tumor treated with the vehicle was used as a control (CNTR). 3B Graph showing the growth rate of LNCaP xenografts in NRG mice after castration (CAST) and intraperitoneal administration of vehicle (n=19 mice), dTRIM24 (n=12 mice), and enzalutamide (ENZA) (n=8 mice). Castration was performed (arrow) when thetumor reached an average size of 50-75 mm3 (indicated by the arrow). The treatment was given for three weeks, five days a week, at 10 mg / kg for dTRIM24 and 30mg / kg for enzalu- tamide. The square marks the treatment period. The two-way ANOVA test was used. 3C Immunoblot of TRIM24, AR, phospho-STAT3, and total STAT3 and Tubulin in tumor relapse after castration, dTRIM24 or vehicle (CNTR). 3D Immunoblot of TRIM24 in LNCAP cells stably transduced with short-hairpin (sh) shSCR (control, CNTR), shTRIM24-l (TRIM24-KD-1), and shTRIM24-2 (TRIM24-KD-2). 3E Growth rate of

[0026] CNTR (n=6 mice), TRIM24-KD-1 (n=8 mice), and TRIM24-KD-2 (n=6 mice) of LNCaP xenografts in NRG mice. Castration (arrow) was performed when the tumor reached an average size of 50-75 mm3. *** P<0.0001; two-way ANOVA test. 3F, UMAP plot of single- cell RNA sequencing data showing the distribution of cell clusters of LNCaP CNTR, TRIM24-KD-1, and TRIM24-KD-2 in residual disease after castration. Out of 9 clusters, 3 are in common (clusters 0, 6, 3), 3 are unique for CNTR (clusters 1, 2, 4), and 4 are unique for TRIM24-KD-1 and TRIM24-KD-2 (clusters 5, 7, 8, 9). 3G Violin plot shows the single- sample gene set enrichment (ssGSEA) score for the progression signature in the cell clusters. The signature was derived by differential expression analysis between pre-castration and relapsed LNCaP xenografts. The top 200 differentially expressed genes (DEGs) based on log2FC were selected to create the signature. The p-value between the clusters (1, 2, 4) and (0, 5, 6, 7, 8, 9) was calculated using the Wilcox test.

[0027] Figure 4. 4A Immunoblot of TRIM24, phospho-STAT3, STAT3, N-Cadherin (N-CAD), E- Cadherin (E-CAD), and Tubulin in LNCaP cells maintained for 3 days in CSS (androgen- free serum) medium alone (CSS, lane 2) or in combination with 1 nM DHT (CSS+DHT, lane 1), or enzalutamide (CSS+ENZA, lane 3) at the indicated concentration. 4B The inter- action of phospho-STAT3 and TRIM24 was assessed by immunoprecipitation (IP) of TRIM24 in LNCaP cells maintained for 3 days in CSS+DHT, CSS, or CSS+ENZA. IP with anti-TRIM24 (lanes 4-6) and anti-IgG control (lanes 1-3). On the right is the immunoblot of TRIM24 and Tubulin of the input lysate. 4C The knock-down or degradation of TRIM24 reduced the activation of STAT3, counteracting EMT in LNCaP. In the left panel, immunoblot of TRIM24, Phospho- STAT3, STAT3, N-Cadherin (N-CAD), and Tubulin in LNCaP cells maintained for 3 days in CSS with enzalutamide (CSS+ENZA, lane 1) alone or in combination with dTRIM24 (CSS+ENZA+dTRIM24, lane 2) at the indicated concentration. In the left panel, immunoblot of the indicated protein in CNTR (lane 1), TRIM24-KD1 (lane 2), and TRIM24-KD2 (lane 3) LNCaP cells maintained for 3 days in CSS medium with enzalutamide. 4D The knock-down or degradation of TRIM24 downmodulated the expression of STAT3-related cytokines induced by enzalutamide treatment. In the right panel, bar plot showing qPCR analysis of TGFP, TNFA, IL-10, IL-6, and IL-ip in LNCaP cells cultured for 3 days in CSS+DHT, CSS+ENZA, and CSS+ENZA+dTRIM24. In the left panel, the corresponding qPCR analysis in CNTR, TRIM24-KD1, and TRIM24-KD2 LNCaP cells maintained for 3 days in CSS+ENZA. The fold change is calculated based on the average expression in the DHT condition (dashed gray line), and the graph reports the media (barplot; SEM) of at least 3 independent experiments. The unpaired Student’s t-test was used. 4E In the left panel, 24-hour trans-well assay of LNCaP cells maintained previously for 3 days in CSS+DHT (CNTR), CSS+ENZA, and CSS+ENZA+dTRIM24. The migrated cells were stained with crystal violet and visualized with a microscope. The bar plot shows the cluster number of migrated cells. In the right panel, the trans-well assay performed with the CNTR, TRIM24-KD1, and TRIM24-KD2 LNCaP cells maintained in CSS+DHT and CSS+ENZA. The bar plot shows the percentage of migrated cells. Each graph reports the media (barplot; SEM) of 4 independent experiments. The unpaired Student’s t-test was used. 4F In the upper panel, wound healing assay by live cell imaging of LNCaP in different medium conditions. LNCaP cells were maintained previously for 3 days in CSS+DHT (CNTR), CSS+ENZA, and CSS+ENZA+dTRIM24, scratched, and then wound closure assessed for 120 hours. The graph shows the relative wound density of LNCaP cells at different time points. In the lower panel, the wound healing assay performed with the CNTR, TRIM24-KD1, and TRIM24- KD2 LNCaP cells in the described condition. Statistical significance is determined using a two-way ANOVA test. It reported a representative experiment of biological triplicate. 4G The TRIM24 degradation reduced activation of STAT3, counteracting EMT in LAPC4 and VCAP cell lines. Immunoblot of the specific protein in the indicated cell line maintained for 3 days in CSS with enzalutamide (CSS+ENZA, lane 1) alone or in combination with dTRIM24 (CSS+ENZA+dTRIM24, lane 2) at the indicated concentration.

[0028] Figure 5. 5 A Immunoblot of TRIM24, AR, E-Caderin (E-CAD), Vimentin, and Tubulin in SPOP-mutant LuCaP147 (line 1) and LuCaP147-CR (castration-resistance) (line 2) xeno- graft tumors in NRG mice in vivo. 5B Immunoblot of TRIM24 and Tubulin of LuCaP147 xenografts following daily intraperitoneal administration of dTRIM24 at 5mg / Kg for five 25 days. Mice treated with the vehicle were used as a control (CNTR). 5C Graph displays the growth rate of LuCaP147 xenografts in NRG mice after castration and treatment with vehicle (CNTR, n=12 mice), and dTRIM24 (n=6 mice). Castration was performed when the tumor reached an average size of 50-75 mm3. dTRIM24 was administered intraperitoneally for three weeks, five days a week, at 5 mg / kg dosage. The square highlights the treatment period. The statistical significance of the results was tested using a two-way ANOVA test. 5D The residual LuCaP147 xenografts after castration were analyzed using single-cell RNA sequencing. The violin plots show the single-sample gene set enrichment (ssGSEA) score for the AR response signature and EMT pre-castration (CAST) and residual vehicle (CNTR) and upon dTRIM24 administration. The statistical significance is calculated using the Wil- con test. 5E Growth rate of LuCaP147-CR xenografts in castrated NRG mice after treatment with vehicle (CNTR; n=8 mice) and dTRIM24 (n=9 mice). The treatment was given intraperitoneally for four weeks, five days a week, at 5 mg / kg. The statistical significance of the results was tested using a two-way ANOVA test. 5F Graph showing the corresponding weight changes.

[0029] Glossary

[0030] As used herein, the expression “pharmaceutical active ingredient” or more simply “active ingredient” refers to any pharmaceutically active molecule (chemical compound, monoclonal antibody, peptide, etc.), for instance, a molecule that can be used for cancer treatment. As used herein, the term “subject” relates to animals, such as mammals, including human beings, cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like. As used herein, the term “treating” or “treatment” refers to evidence of success or improvement in the treatment of a certain disease, lesion, condition, or symptom, or, in certain cir- cumstances, the prevention of the onset of a symptom or condition. As used herein, "prevent", "preventing", "prevention", or "prophylaxis" of a disease or dis- order means preventing that such a disease or disorder occurs in a patient, such as the relapse after treatment. In any point of the present specification or in the claims, the terms “comprising” or “com- prise(s)” can be replaced by the terms “consisting of’ or “consist(s) of’.

[0031] As used herein, the term “Degron” serves to link a targeted protein, through a Linker and a Targeting Ligand, to a ubiquitin ligase for proteosomal degradation. In one embodiment, the Degron is capable of binding to a ubiquitin ligase, such as an E3 ubiquitin ligase. In one embodiment, the Degron is capable of binding to cereblon. The terms “disease(s)”“disorder(s)”, and “ condi tion(s)” are used interchangeably, unless the context clearly dictates otherwise.

[0032] AR-V7 refers to a ligand-independent and constitutively active variant of the AR. AR-V7 and other ligand-indpendent isoforms of AR are considered key drivers of ARSi resistance in CRPC.

[0033] Detailed description of the invention

[0034] Prostate cancer typically progresses from hormone-sensitive to castration-resistant disease.

[0035] The invention shows that the pharmacological degradation of tripartite motif-containing protein 24 (TRIM24) effectively inhibits castration-resistant prostate cancer growth in vivo and may be used for the treatment thereof. Second, the degradation of TRIM24 counteracts resistance mechanisms actively induced by androgen deprivation therapy and androgen receptor signaling inhibitors (ARSi) or any other type of AR inhibition (e.g., pharmacological AR degradation) (e.g. STAT3, EMT) and thereby in combination with the latter prevents or delays tumor relapse and progression to castration-resistant disease. Third, the effect of TRIM24 degradation is particularly effective in prostate cancer characterized by recurrent point mutations in the ubiquitin ligase adaptor SPOP in any setting. In one embodiment the present invention refers to a compound capable of inhibiting TRIM24 activity for use in combination with androgen deprivation therapy (ADT) and / or androgen receptor signaling inhibitors (ARSi) or any other type of AR inhibition (e.g., pharmacological AR degradation) in a method of treatment of a hormone-sensitive metastatic prostate cancer and / or in preventing or delaying tumor relapse and progression to castration-resistant disease. The treatment with said TRIM24 inhibitor may start before or at the time point the androgen deprivation therapy and / or androgen receptor signaling inhibitors or any other type of AR inhibition (e.g., pharmacological AR degradation) start.

[0036] Suitable androgen receptor signaling inhibitor in said combination are for example enzalutamide, darolutamide, apalutamide, or abiraterone.

[0037] Suitable androgen deprivation therapy consists for example in the administration of leuprorelin (leuprolide), goserelin, triptorelin, histrelin, buserelin and / or degarelix. Suitable pharmacological AR degraders are in clinical development, such ARV-110 or ARV-766. In one preferred embodiment the subject treated with said compound capable of inhibiting TRIM24 activity is a subj ect with a prostate cancer characterized by recurrent point mutations in SPOP. The present invention refers also to a compound capable of inhibiting TRIM24 activity for use in a method of a treatment of a castration-resistant prostate cancer, most notably driven by AR-V7 or other AR isoforms lacking the ligand binding domain. In one preferred embodiment said prostate cancer is characterized by recurrent point mutations in SPOP. Given the similarities of sex steroid hormone signaling in luminal breast cancer (estrogen receptor) and prostate cancer (androgen receptor) and the prior publications suggesting an oncogenic effect of TRIM24 in breast cancer, it is very likely that pharmacologic degradation of TRIM24 may be also effective in advanced endocrine-resistant breast cancer and may synergize with endocrine therapies (e.g., selective estrogen receptor modulators such as tamoxifen, aromatase inhibitors such as letrozole, selective estrogen receptor degraders such as fulvestrant, pharmacological ER degrader such as ARV-471) in preventing disease progression. Accordingly, in one embodiment the present invention refers to a compound capable of inhibiting TRIM24 activity for use in a method of treatment of advanced endocrine-resistant breast cancer, in particular in combination with endocrine therapies, such for example the selective estrogen receptor modulators tamoxifen. A further object of the present invention is a pharmaceutical composition comprising a compound capable of inhibiting TRIM24 activity and a pharmaceutically acceptable carrier for any use herein disclosed. In one embodiment the pharmaceutical composition further comprises an androgen receptor signaling inhibitor such for example enzalutamide, darolutamide, apalutamide, abiraterone, and / or androgen deprivation therapy compounds such for example leuprorelin (leuprolide), goserelin, triptorelin, histrelin, buserelin and / or degarelix and / or pharmacological AR degraders, for example AR degraders that are in clinical development, such ARV-110 or ARV-766. The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound capable of inhibiting TRIM24 activity, or an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier for use in any treatment herein disclosed. In another aspect, the application provides a kit comprising a compound capable of inhibiting TRIM24 activity selected from one or more compounds disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, optionally in combination with a second agent and instructions for use in any treatment herein disclosed. The compound capable of inhibiting TRIM24 activity can be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g., orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injectable solutions or suspensions, or topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal or suppository form. For example, oral compositions can be tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and / or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and / or e) absorbents, colorants, flavors and sweeteners. Injectable compositions can be aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions. As used herein, the term “pharmaceutically acceptable carrier” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of this application can be administered to humans and other animals orally, rectally, parenterally, intraci sternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), buccally, or as an oral or nasal spray. The compound capable of inhibiting TRIM24 activity means any molecule that decreases the TRIM24 activity. The potency of the inhibitor can be determined for example by EC50 value. A compound with a lower EC50 value, as determined under substantially similar conditions, is a more potent inhibitor relative to a compound with a higher EC50 value. In some embodiments, the substantially similar conditions comprise determining a TRIM24- dependent phosphorylation level, in vitro or in vivo (e.g., in cells expressing a wild-type TRIM24, a mutant TRIM24, or a fragment of any thereof). Potency of the inhibitor can also be deter- mined by IC50 value. A compound with a lower IC50 value, as determined under substantially similar conditions, is a more potent inhibitor relative to a compound with a higher IC50 value. In some embodiments, the substantially similar conditions comprise determining a TRIM24-dependent phosphorylation level, in vitro or in vivo (e.g., in cells expressing a wild- type TRIM24, a mutant TRIM24, or a fragment of any thereof). In one preferred embodiment, the compound capable of inhibiting TRIM24 activity is a compound that target TRIM24 for degradation. Some embodiments relate to the loss of 50-100% of the target protein TRIM24. Other embodiments relate to the loss of 75-95% of the targeted protein TRIM24.

[0038] In one preferred embodiment said TRIM24 inhibitors may include bifunctional compound or molecular glue capable of degrading the targeted protein TRIM24 through the UPP pathway. The difference between the molecular glue and the bifunctional compound is that they consist only of one small molecule and not two molecules linked with a linker.

[0039] In one embodiment said TRIM24 inhibitors are used also as (neo)-adjuvant treatment in combination with AR inhibition (e.g., ADT + ARSi) and radiotherapy or surgery in the treatment of the primary tumor. Bifunctional compounds that target TRIM24 for degradation suitable for use in the present invention are for example the bifunctional compound disclosed in US10702504 (herein incorporated by reference) or other bifunctional compounds capable of degrading a targeted protein TRIM24 through the UPP pathway. Accordingly, the bifunctional compound of the present application is any of the formulae disclosed in US10702504 (herein incorporated in its entirety by reference). The synthesis of the bifunctional compounds can be found in US 10702504. Without binding the present invention to any theories, the following molecular mechanism is disclosed: CRPC: During disease progression under ADT / ARSi prostate cancer cells adapt to the low levels of androgens through upregulation of TRIM24 which helps to recruit AR to genes important for cell proliferation and cancer cell survival. Because the activation of AR signaling does not directly involving androgens, targeting these mechanisms may trigger substantial anti-tumor responses when ADT / ARSi does not work anymore. Synergy with ADT / ARSi: The synergy between dTRIM24 and ADT / ARSi is explained by the fact that dTRIM24 inhibits AR signaling (see above) and additional pathways (e.g., STAT3, EMT) which are triggered directly by ADT / ARSi and have been shown to contribute to resistance development. SPOP mutant prostate cancer: Recurrent mutations in SPOP disable directly ubiquitylation and degradation of TRIM24 8,11. Cell culture evidence suggests that this upregulation con- tributes to the oncogenic effect of these mutations 8. Thus, TRIM24 may be a critical target in this genetically defined subtype of prostate cancer. Also, the method of treatments of a subject in the need thereof is also disclosed.

[0040] In the present application are also disclosed methods of treatments according to any definition herein provided comprising the step of the administration of a compound capable of inhibiting TRIM24 activity according to any embodiments herein disclosed in a patient.

[0041] The following examples are provided for illustrative purposes and not as a limitation of the scope of the invention as defined in the appended claims.

[0042] EXAMPLES

[0043] Example 1

[0044] MATERIAL & METHODS Plasmids

[0045] The pLKO-SCR (SHCC002Sigma-Aldrich), the pLKO-shTRIM24-l (TRCN0000195528, Sigma-Aldrich), and the PLK-shTRIM24-2 (TRCN0000021259, Sigma-Aldrich) were pur- chased from Sigma.

[0046] Cell Lines

[0047] LNCaP LAPC4, VCaP, 22RV1, and HEK 293T cell lines were purchased from ATCC (American Tissue Culture Collection) (Manassas, USA). The LNCaP-95 cell line was a gift from Prof. Myles Brown (DFCI, Boston. The supernatant of all cell lines was routinely tested (once per month) using the MycoAlertTM Mycoplasma Detection Kit (Catalog #: LT07-318 Lonza). All cell lines resulted negative for Mycoplasma infection.

[0048] Cell Culture

[0049] The LNCaP, and the 22RVlcell line was cultured in RPMI 1640 medium (Cat 21875-24, Gibco) supplemented with 10% Fetal Bovine Serum (FBS-11A Capricorn Scientific) and 1% Penicillin / Streptomycin (15140-122 Life Technologies) with 5% CO2 at 37°C. The LAPC4 cell line was cultured in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum, InM DHT (5α-Dihydrotestosterone, D-073, Sigma-Alderich), and 1% Penicillin / Streptomycin, with 5% CO2 at 37 °C. The LNCaP-95 cell line was cultured in Phenol-Red Free RPMI 1640 medium (11835-063 Gibo) containing 10% charcoal-stripped serum (CSS Fetal Bovine Serum, Charcoal Stripped; Cat 12676-029 Gibco) and 1% Penicillin / Streptomycin with 5% CO2 at 37°C. The HEK 293T, and the VCAP cell line was cultured in DMEM medium (Cat 2688-133, Gibco) supplemented with 10% FBS and 1% μg / ml Penicillin / Streptomycin with 5% CO2 at 37°C.

[0050] PEI-mediated transfection and Lentiviral Infection

[0051] The HEK 293 T cells were transfected with a vector for each shRNA, packaging, and enve- lope vector using the PolyEthylenlmine (PEI) method. The HEK 293 T cells were seeded in a 100 mm culture dish (4× 10Λ6 cells / plate) and incubated overnight at 37°C in a 5% CO2 humidified atmosphere. After 24 hours, the vector plasmid (3 μg), packaging plasmid (pCMV-dR8.2; 2.7 μg), and envelope plasmid (pVSV-G; 0.7 μg) were mixed in Opti- MEM™ I Reduced Serum Medium (300 μL / 10 cm culture dish; Cat 31985-070, Gibco) andwith 1.25 mM PEI (Sigma-Aldrich 919012) solution (ratio pL PEI: μg DNA 4: 1). The DNA / PEI mixture were incubated for 15 minutes at room temperature and added to the HEK 293 T cell supernatant. After 48 hours of transfection, the viral supernatants were collecte and filtered through a 0.45μm filter. The LNCaP, LNCaP-95, and 22RV1 cell lines were incubated with viral supernatant and 8 μg / ml of Polybrene (H9268, Sigma- Aldrich) for 72 hours. After incubation, the selection was performed using a specific antibiotic for a minimum of 10 days: 2 μg / ml of puromycin (P8833, Sigma-Aldrich) for cells infected with the PLKO vectors or 10 μg / ml of blasticidin (15205, Sigma-Aldrich) cells infected with the PLV vectors.

[0052] Tumor Tissue Enzymatic Digestion

[0053] We followed a specific procedure to obtain a single-cell suspension of tumor cells from pa- tient-derived xenograft (PDX) tumor tissue. First, we cut the tumor tissue into small pieces (1-0.5 mm) using a scalpel blade. Then, we digested the tissue for 45-60 minutes in a DMEM / F-12, GlutaMAX™ medium (Cat 31331-028, Gibco) with Collagenase Type I (200U / ml; Cat 17018-029, Gibco) and DNAsel (Img / ml; Cat 11284932001, Roche) at 37°C. After enzymatic dissociation, we filtered the cell suspension through a 100 μM cell strainer (Roche) to eliminate macroscopic tissue pieces and then centrifuged it. The resulting cell pellet was resuspended in a 2-volume RBC lysis buffer (Roche) and incubated for 3 minutes at room temperature. After centrifugation, the cells were resuspended in a PBS solution. We used this single-cell suspension to inject animals for pharmacological studies mentioned in the animal section.

[0054] Colony Formation Assay

[0055] The LNCaP, the LAPC4, 22RV1, and LNCaP-95 parental cell lines were seeded in duplicate into 12-well plates at a density of 12500 cells per well. Before the seeding the 12-well plates were pre-coated with a solution of poly-D lysine (120 μg / mL in PBS) for 24 hours at 37°C. All cell lines were cultured in a testosterone-depleted medium (charcoal-stripped serum; CSS) supplemented with the following conditions: (i) 1 nM DHT alone or in combination with 5 μM of dTRIM24-l or 5 μM of dTRIM24-2; (ii) without DHT, either alone or in combination with 5 μM of dTRIM24-l or 5 μM of dTRIM24-2; (iii) without DHT, supplemented with 10 μM of enzalutamide alone or in combination with 5 μM of dTRIM24- 1 or 5 μM of dTRIM24-2. The medium was refreshed 3 times a week. After 18-20 days, colony confluence was assessed using the IncuCyte live-cell analysis system. The LNCaP, 22RV1, and LNCaP-95 cells infected with control, shTRIM24-l, and shTRIM24-2 vectors were examined under the same conditions. The experiments with VCaP cells were performed using 1 μM of dTRIM24-l or 1 μM of dTRIM24-2 instead of 5 μM and maintained a minimum concentration of 0.01 nM DHT in a testosterone-depleted condition. Each cell line and condition were tested in biological triplicate. Transwell Assay

[0056] LNCaP parental cells and LNCaP stable transfected with control, shTRIM4-1 , and shTRIM24-2 pLKO vectors were cultured in a testosterone-depleted medium (charcoal- stripped serum - CSS) with lOuM of Enzalutamide alone or with or 5uM of dTRIM24 (in the case of parental cells) for 3 days. The LNCaP cells cultured in CSS supplemented with InM DHT were used as a control group. After three days, 5 x 10A4 serum-starved cells were added to the upper compartment of the Boyden chamber, and the lower compartment was filled with RPMI medium containing 10% FBS. After 24 hours of incubation, the percentage of invaded cells was measured. The migrated cells were stained with a 2% crystal violet staining solution and visualized under a micro- scope.

[0057] Incucyte® Scratch Wound Analysis

[0058] A wound healing assay was conducted on LNCaP parental cells or LNCaP stable transfected with control, shTRIM4-1 , and shTRIM24-2 pLKO vectors using live cell imaging on the In- cuCyte system. The cells were cultured in a 96-well plate with different medium conditions. Specifically, LNCaP cells at a density of 50000 cells / well were maintained in a testosterone- depleted medium (charcoal-stripped serum - CSS) supplemented with either lOuM of Enzalutamide alone or 5uM of dTRIM24 (in the case of parental cells). The control group consisted of LNCaP cells cultured in CSS supplemented with InM DHT. After three days of culture, the feeder layer of cells was scratched, and then the cells were tested for 120 hours. The IncuCyte® software was

[0059] used to measure the spatial cell density in the wound area relative to the spatial cell density outside the wound.

[0060] Antibodies per Immunoblot and immunoprecipitation

[0061] The primary antibodies used are anti-Tubulin (3873 Cell Signaling Technology), anti-AR (133273 abeam), anti-TRIM24 (14208-1-AP Proteintech), anti-GAPDH (sc-47724, Santa Cruz Biotechnology), anti-NKX3.1 (87300, Cell Signaling Technology), anti-E-Caderin (3195 Cell Sig- naling Technology), anti-Vimentin (5741 Cell Signaling Technology), anti- N-cadherin (13116 Cell Signaling Technology), anti-STAT3 (8019 Cell Signaling Technology), and anti-Phospho-STAT3(Ser727) (9134 Cell Signaling Technology). Snap- frozen tumor tissue (Fragments of 25-30 mg) or cellular pellet was lysed using RIPA Buffer supplemented with a cocktail of phosphatase inhibitors (4906845001 Roche) and protease inhibitors (5892953001 Roche). The protein concentration was determined using a BCA reagent (A52255 Thermo Fisher Scientific). 30-50 μg of whole protein lysate was separated on 8- 12% SDS-polyacrylamide gels and transferred onto PVDF membrane (88518 Thermo Fisher Scientific). The membranes were first blocked with 5% milk in Tris Buffered Saline with Tween 20 (TBST) for 30 minutes at room temperature. Only for the phospho- STAT3 antibody the blocking was made in 5% BSA. After that, they were incubated with primary antibodies overnight at 4°C. Then, they were incubated with secondary antibodies (anti-rab- bit IgG HRP W401B and anti-mouse IgG HRP W402B Promega) for 1 hour at room temperature. The protein bands were visualized using the western bright quantum reagent (K- 12042-D20 Advansta) and quantified using the Fusion Solo IV LBR system.

[0062] Immunoprecipitation protocol

[0063] Immunoprecipitation (IP) analysis was conducted on LNcaP cells cultured in CSS mediumfor 3 days. The cells were treated with either DHT InM or Enzalutamide lOmicroM. The IP Buffer, (20 mM Tris-HCl (pH 7.5), 150mM NaCl, ImM EDTA, 2mM Na3VO4, 5mM NaF, and 1% Triton X-100, protease inhibitor cocktail, and phosphatase inhibitor cocktail), was used to lysate the cellular pellet. The anti-TRIM24 (3 micrograms) antibodies were incu- bated with Img of cell lysates overnight. Anti-IgG was used as a control. The samples were immunoprecipitated by using the Dynabeads Protein A (Cat 1000 ID, Invitrogen) immuno- precipitated the samples. The immunoprecipitates were washed with the IP buffer thrice, resolved in a 2x SDS lysis buffer, and analyzed in an SDS-PAGE gel. RT-qPCR analysis

[0064] RNA was extracted from a cellular pellet of LNCaP cells using an RNeasy kit (74106 Qi- agen), following the manufacturer's guidelines. The extraction was performed at the indicated culture condition. RT-qPCR analysis was carried out using KAPA SYBR® FAST One-Step (KK4600 Sigma) per the manufacturer's protocol. The primer sequences were ob- tained from PrimerBank (http: / / μga.mgh.harvard.edu / primerbank / index.html), see Table 1 below. The housekeeper gene used was Actin. The qPCR analysis was performed using the 2-AACt method.

[0065] RNA Extraction

[0066] RNA extraction was performed from snap-frozen tumor fragments (25-30 mg) of cellular pellets using the RNeasy kit from Qiagen (74104) following the manufacturer's instructions. For bulk RNAseq analysis, the extracted RNAs were processed using the NEB Next Ultra II Directional Library Prep Kit for Illumina and then sequenced on the Illumina NextSeq500 with single-end, 75-base pair-long reads. scRNAseq analysis

[0067] To perform scRNA-seq, we isolated a single-cell suspension of cells from freshly isolated tumor fragments (see Tumor Tissue Enzymatic Digestion section). We resuspended the sin- gle-cell suspensions in PBS and loaded them into a lOx Chromium Controller from lOx Genomics (located in Pleasanton, CA, USA), aiming to obtain 10000-5000 cells. We used the Chromium Next GEM Single Cell 3' v3.1 reagent kit from lOx Genomics (PN-1000121) and followed the manufacturer's instructions.

[0068] Animal Experiments

[0069] All animal experiments were conducted following a protocol that was approved by the Swiss Veterinary Authority / Board (TI-40-2018, TI 41-18, TI-44-2019 TI30-21 TI-49-2023) and received approval from the ethical committee of the Institute of Oncology Research (IOR). For all in vivo studies, we used male NRG (NOD-Raglnull IL2rgnull, NOD rag gamma) mice between 6-8 weeks old. Manage and generate the Patient-Derived Xenograft (PDX) line.

[0070] The LuCaP-147 patient-derived xenograft (PDX) was provided by Dr. Eva Corey. This PDX line is maintained in vivo by subcutaneously transplanting Matrigel-embedded tissue tumor fragments into male NRG mice. To create CR LuCaP-147 sublines, we harvested tissue fragments from a LuCaP-147 (relapsed) tumor that regrew after castration. These CR LuCaP-147 lines are then passed on to other castrated male mice and designated as CR after being passed on at least three times.

[0071] In vivo pharmacology studies

[0072] For the in vivo studies of drug treatment in combination with surgical castration, we used LNCaP cell lines, or freshly isolated single-cell suspensions from the LuCaP-147 tumor fragment (see Tumor Tissue Enzymatic Digestion session). To start the study, we suspended the tumor single cells in PBS and 50% Matrigel and injected it subcutaneously into the dorsal flanks of the mice. We used 2.5 x 10A6 cells / mouse for LNCaP, and 1 x 10A6 cells / mouse for LUCaP-147. We recorded tumor growth using a digital caliper and calculated tumor vol- umes using the formula (L x WA2) / 2, where L is the length, and W is the width of the tumor. Tumor volume was measured twice a week. Once the tumor dimension reached 50-75 mmA3, we surgically castrated the mice. The treatment was given after castration, intraperi- toneally, for three weeks, five days per week. For LNCaP we used 10 mg / kg for dTRIM24-l-2, and 30mg / kg for Enzalutamide for the LuCaP-147 PDX line, we used 5 mg / kg for dTRIM24. At the end of the experiment, we euthanized the mice, explanted the tumors, and used them for molecular assessment.

[0073] We used LNCaP-95 cells or freshly isolated single-cell suspensions from the LuCaP-147- CR tumor fragment for the studies on castrated NRG mice. We inj ected 1 x 10A6 cells / mouse for LNCaP-95 and 1 x 10A6 cells / mouse for LUCaP-147 in PBS and 50% Matrigel subcutaneously into the dorsal flanks of the mice. The drug treatment started when the tumor size grew to 25-50 mmA3. For LNCaP-95, we administered 10 mg / kg of dTRIM24 and 30mg / kg of Enzalutamide intraperitoneally for two weeks. For the LuCaP-147 PDX line, we used 5 mg / kg of dTRIM24 intraperitoneally for three weeks, five days a week. After the treatment, we euthanized the mice and removed the tumors for molecular assessment.

[0074] Statistical analysis

[0075] The data was analyzed using GraphPad Prism. The means ± standard errors are presented in the results. Student t-tests were used to identify differences between the two groups. For multiple groups, one-way analysis of variance (ANOVA) tests were used. Statistical analyses were based on data collected from at least three independent experiments. P<0.05 indicated statistically significant differences.

[0076] Bulk RNA-sequencing Analysis

[0077] Quantification of gene expression

[0078] The overall quality of sequencing reads was evaluated using FastQC (v.0.11.9). Sequence alignments to the reference human genome (GRCh38.pl3) were performed using STAR (v.2.7.1b) in two-pass mode, to significantly increase sensitivity to novel splice junctions compared to the regular single mapping. In the two-pass mapping procedure, reads are mapped twice: in the first pass, the novel junctions are detected and inserted into the genome indices; in the second pass, all reads are re-mapped using annotated (from the GTF file) and novel (detected in the first pass) junctions. Gene expression was quantified at the gene level in the second pass by using the comprehensive annotations made available by Gencode (v37 GTF File). Strand-specific information was not maintained to avoid technical differences between stranded and unstranded libraries.

[0079] Samples were adjusted for library size and normalized with the variance stabilizing transformation (vst) in the R statistical environment using DESeq2 (vl .34.0) pipeline. When per- forming differential expression analysis between groups, we applied the embedded Inde- pendentFiltering procedure to exclude genes that were not expressed at appreciable levels in most of the samples considered.

[0080] Gene-set enrichment analysis

[0081] All GSEAs were performed using Custerprofiler(v4.2.2) package (GSEA, eps=le-50). Gene-set collections were retrieved from the Molecular Signature Database (MSigDB) or previous publications (AR / NE- Score). P values were corrected for multiple testing using the false discovery rate (FDR) procedure, with the significance threshold set to 0.05. In addition, GSEA significance was logarithmically transformed in the form of -10log10(p-adjusted), with a bold intercept (x = 13.01) indicating the FDR threshold depicted in the corresponding plots. Over-representation analyses (ORA) were performed using Custerprofilerc(v4.2.2) package (enrichR).

[0082] Single-cell RNA-sequencing

[0083] Quantification of gene expression

[0084] Fastq files were generated by demultiplexing raw data using Cellranger mkref (v7.1.0). We generated a custom genome with Cellranger, using the same reference (GRCh38.p13) and annotations (Gencode v37) we used for STAR when performing bulk RNA-seq analysis. To discriminate between human and murine cells that may infiltrate the tumors in the in vivo setting, we created a Mouse-Human reference, by creating a hybrid genome (GRCh38.p13 + GRCm39) and hybrid gene-annotations (Gencode v37 and M26, for human and mouse genes, respectively). Such reference has been used for reads alignment, per- formed using Cellranger. To avoid conflicts, a suffix has been added to mouse genomic co- ordinates (i.e., chr1M, chr2M, etc.). Subsequently, Cellranger was used to quantify gene expression in the form of an h5-filtered matrix where Ensembl gene IDs were used as iden- tifiers.

[0085] Data filtering and clustering

[0086] Expression quantification files were imported into R statistical environment using Seurat (4.3.0) package. Individual cells from the data matrix were discarded by using a two-filtering procedure: first, we aimed at detecting transcriptional outliers, and second, we looked for putative doublets, which we also discarded. Briefly, we computed per-cell quality control metrics using scater (vl.22.0). The total amount of mitochondrial and ribosomal gene expression was quantified for both human and mouse cells. The number of genes being detected per cell, the total amount of reads per cell, and the mitochondrial and ribosomal fraction of the transcriptome were used to determine the skewness-adjusted multivariate out lyingness for each cell (robustbase v0.95-0). Outliers were detected by median absolute deviation and removed at both tails. Counts were then normalized (Seurat: :NormalizeData, method = LogNormalize, scale. fac- tor = 1000) and the top 2000 most variable features were selected (Seurat: :FindVariable- Features, method = vst). Data were then scaled (Seurat: : ScaleData) and PCA was performed up to the top 50 components (Seurat: :RunPCA). Subsequently, we identified and eliminated putative doublets using DoubletFinder (v2.0.3). Having identified outliers and doublets, we removed them from the original count data and repeated the preprocessing step (i.e., normalization, scaling, and dimensionality reduction). We then proceeded to determine the k-nearest neighbors of each cell and the construction of a shared nearest-neighbor (SNN) graph (Seurat: :FindNeighbors), then we identified clusters using the SNN modularity optimization-based clustering algorithm (Seurat: :FindClusters, resolution = 0.5). Finally, we performed Umap dimensionality reduction on the first 30 PCs, annotated the previously identified clusters, and generated plots accordingly.

[0087] Dealing with drop-out events

[0088] Drop-out events are very frequent in the single-cell experiment performed using lOx Chro- mium technology. When drop-out occurs, the absence of data for a particular gene in a spe- cific cell can introduce biases and distort the overall picture of gene expression patterns. This can lead to incorrect interpretations of the data, especially when trying to identify rare cell types or subtle differences between cells. To address these issues, we applied Markov Affinity-based Graph Imputation of Cells (MAGIC algorithm, RMagic v2.0.3) Differential expression analysis and gene-set enrichment.

[0089] Differential expression was performed between cell clusters subjected to different treatment, conditions (Seurat: :Findmarkers) using a hurdle model tailored to scRNA-seq data (MAST method). Genes were subsequently ranked for log2 FC, and the Camera algorithm (pre- ranked) was used to determine gene-set enrichments for each comparison.

[0090] Cell-specific gene-set enrichments were determined using single-sample GSEA (Seu- rat: :AddModule Score), computed using gene-expression values of each cell following RMagic imputation.

[0091] Identification of cell type Murine cells could be clearly distinguished from human cancer cells, because of the intrinsic differences that could be easily spotted owing to the alignment and quantification performed using a hybrid human-mouse genome. Murine cell types were identified using SingleR (vl.8.1), using consensus from the ImmGen repository and the signatures from Benayoun, B. et al, 2019.

[0092] Gene signatures

[0093] The gene signatures utilized are derived primarily from the Molecular Signature Database (MSigDB) and the scientific literature. The Molecular Signature Database provides a com- prehensive collection of gene sets that represent various biological processes, pathways, and functional annotations. Specifically, it includes hallmark gene sets (H), which capture fun- damental biological processes and signaling pathways that are commonly dysregulated in different diseases or conditions. Additionally, the curated gene sets in MSigDB (C2) encom- pass specific biological knowledge curated from diverse sources. Also, the Gene Ontology database (C5) has been used to enrich for specific biological processes or molecular func- tions. The Progression Signature was derived from single-cell RNA-sequencing data after differential expression analysis (Seurat: :FindMarkers, method=MAST) between the re- growth condition and the pre-castration sample. Differentially expressed genes were ordered by log2FC and the first 200 hints were used to create the signature.

[0094] EXAMPLE 2 dTRIM24 inhibits the in vivo growth of the CPRC model LNCaP-95 (Fig. 1): LNCaP - 95 has been derived from parental LNCaP cells through chronic androgen deprivation and represents a model for CPRC. dTRIM24 diminishes TRIM24 protein levels in culture and in vivo (Fig. 1 A, B). Daily intraperitoneal (i.p.) treatment of LNCaP-95 xenografts with dTRIM24 (5 times a week) significantly diminishes tumor growth while enzalutamide has little effect (Fig. 1C). dTRIM24 is well tolerated at a concentration of lOmg / kg in NRG mice and no weight loss (Fig. 1D). Gene set enrichment analysis of RNA sequencing data of control versus dTRIM24 in culture shows loss of androgen response, cell cycle targets (e. MYC), and mTOR signaling (Fig. 1E). Corresponding immunoblot analysis (Fig. 1F) showing suppression of MYC expression, androgen signaling (e.g., reduced PSA expression), and mTOR signaling (phosphorylation of S6K and 4-EBP1). dTRIM24 delays tumor regrowth in vivo after ADT (surgical castration) (Fig. 2): dTRIM24 diminishes TRIM24 protein levels in culture and in vivo of LNCaP cells (Fig. 2A, B). Castration induces tumor shrinkage and relapse after more than 100 days. Treatment with dTRIM24 for 3 weeks (5 times a week) subsequently delays relapse more than enzalutamide administered for the same period (Fig. 2C). Relapsing tumors after dTRIM24 express higher levels of TRIM24 but lower levels of active, phosphorylated STAT3, indicating that insufficient degradation of TRIM24 enabled the survival of tumor cells with higher level of TRIM24 while dTRIM24 was sufficient to reduce STAT3 activation even at the timepoint of tumor relapse off-treatment (Fig. 2D, E). In corresponding experiments with knockdown of TRIM24 using two stable hairpin RNAs (TRIM24-KD1, -KD2), tumor growth is not ma- jorly affected (Fig. 2F, G). Upon castration, tumor relapse is disabled by TRIM24 knock- down. Corresponding single-cell analysis at the level of residual disease shows that upon TRIM24 knockdown cell cluster 1, 2, 4 are eliminated (Fig. 2H). The latter clusters are likely required for tumor relapse as they resemble at the gene expression level best the tumor re- lapse based on a progression signature culled from relapse versus pre-castration (Fig. 21). ADT / enzalutamide activates STAT3 and EMT in LNCaP cells through TRIM24 (Fig.3): Removal of dihydrotestosterone (DHT) and the addition of enzalutamide (ENZA) in an- drogen-depleted medium (charcoal-stripped serum, CSS) increases STAT3 phosphorlyation and engages changes of EMT (E-CAD down, N-CAD up). The absence of DHT and the presence of ENZA further increase STAT3 binding to TRIM24 in immunoprecipiation as- says (Fig. 3B). Conversely, dTRIM24 reduces STAT3 phosphorlyation (Fig. 3C) and reduces the expression of cytokines associated with STAT3 signaling and EMT (Fig. 3D). Finally, dTRIM24 prevents the increases in cancer cell invasion and migration mediated by ADT / ENZA (Fig. 3E, F). The latter processes are both known to be associated with STAT3 and EMT. dTRIM24 strongly synergizes with ADT in preventing tumor relapse of SPOP- Y83C-mutant LuCaP-147 (Fig. 4): LuCaP-147 xenografts relapse in NRG mice with a latency of around 80 days after castration and show EMT as evidenced by AR and E-CAD loss and increase in VIM (Fig. 4A, B). Daily intraperitoneal administration of dTRIM24 at t timepoint of castration for 3 weeks (5 days a week) largely abolishes tumor regrowth (Fig. 4B, C). Single-cell RNA sequencing analysis of residual disease reveals a downregulation of AR, mTOR, and EMT upon dTRIM24 treatment (Fig. 4D). Serial propagation of castration-resistant LuCaP-147CR emerging from the experiment in Fig. 4C reveals continuous activity of dTRIM24 (Fig. 4E). No weight loss is observed in NRG mice harboring LuCaP- 147 xenograft upon dTRIM24 treatment.

[0095] Abbreviations used in the description:

[0096] Androgen receptor (AR)

[0097] Androgen deprivation therapy (ADT)

[0098] Androgen signaling inhibitors (ARSi) Bifunctional TRIM24 degrader (dTRIM24) Charcoal-stripped serum (CSS)

[0099] Castration-resistant prostate cancer (CRPC) Epithelial-to-mesenchymal transition (EMT) Estrogen receptor alpha (ER) Intraperitoneal (i.p.) Signal transducer and activator of transcription 3 (STAT3) Speckle Type BTB / POZ Protein (SPOP) Tripartite motif-containing protein (TRIM) AR splice variant 7 (AR-V7).

Claims

CLAIMS1. A compound capable of inhibiting TRIM24 activity for use in combination with andro- gen deprivation therapy and / or androgen receptor signaling inhibitors and / or pharmacological de- grader of AR or any AR inhibitor in a method of treatment of a hormone-sensitive metastatic pros- tate cancer, optionally in combination with radiotherapy.

2. The compound for use according to claim 1 for preventing or delaying tumor relapse and progression to castration-resistant disease.

3. The compound for use according to claim 1 or 2, where said prostate cancer is characterized by recurrent point mutations in SPOP.

4. The compound for use according to any one of claims 1 to 3, wherein the treatment with said TRIM24 inhibitor started before or at the time point the androgen deprivation therapy and / or androgen receptor signaling inhibitors started or thereafter but before the onset of relapse and / or in a neo-adjuvant setting before surgery of the primary tumor.

5. The compound for use according to any one of claims 1 to 4, wherein said combination therapy comprises a further chemotherapeutic drug, such as docetaxel.

6. The compound for use according to any one of claims 1 to 5, wherein the androgen receptor signaling inhibitor in said combination is enzalutamide, darolutamide, apalutamide and / or abi- raterone.

7. The compound for use according to any one of claims 1 to 6, wherein androgen deprivation therapy consists in the admistration of leuprorelin (leuprolide), goserelin, triptorelin, histrelin, buserelin and / or degarelix.

8. A compound capable of inhibiting TRIM24 activity for use in a method of treatment of a castration-resistant prostate cancer, preferably for use in a method of treatment of castration- resistant prostate cancer expressing AR-V7 or other AR isoforms lacking the ligand-binding domain.

9. A compound capable of inhibiting TRIM24 activity for use in a method of treatment of a patient with castration-resistant prostate cancer, wherein the cancer of said patient is positive for the expression of AR-V7 protein or other AR isoforms lacking the ligand-binding domain.

10. A compound capable of inhibiting TRIM24 activity for use in combination with AR inhi- bition therapy in a method of treatment of a hormone-sensitive metastatic prostate cancer, through an alternative mechanism of AR inhibition.

11. A compound capable of inhibiting TRIM24 activity for use in a method of treatment of anendocrine-sensitive and resistant breast cancer.

12. The compound for use according to any one of claims 1 to 11, wherein said compound ca- pable of inhibiting TRIM24 activity is a compound that target TRIM24 for degradation, in particular is a bifunctional compound or molecular glue capable of degrading the targeted protein TRIM24 through the UPP pathway.

13. The compound for use according to any one of claims 1 to 11, wherein said compound ca- pable of inhibiting TRIM24 activity is selected from a bifunctional molecule of formula X:wherein: the Targeting Ligand is capable of binding to TRIM24; the Linker is a group that covalently binds to the Targeting Ligand and the Degron; and the Degron is capable of binding to a ubiquitin ligase, wherein the Targeting Ligand is of Formula TL-I:or an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt thereof, wherein:A is phenyl or 5- or 6-membered heteroaryl containing 1 or 2 heteroatoms selected from N and S, wherein the phenyl or heteroaryl is optionally substituted with 1 to 3 R 5 ;G is phenyl or 5- or 6-membered heteroaryl containing 1 or 2 heteroatoms selected from N and S, wherein the phenyl or heteroaryl is optionally substituted with 1 to 3 R 6 ;X is S(O) t ;Y is O or NR 4;R1 is H, (C 1 -C 4 ) alkyl, or (C 1 -C 4 ) haloalkyl;R2 is H, (C 1 -C 4 ) alkyl, or (C 1 -C 4 ) haloalkyl;R3 is H, (C 1 -C 4 ) alkyl, or (C 1 -C 4 ) haloalkyl;R4 is H, (C 1 -C 4 ) alkyl, or (C 1 -C 4 ) haloalkyl; each R 5 is independently (C 1 -C 6 ) alkyl, (C 1 -C 6 ) haloalkyl, (C 1 -C 6 ) alkoxy, (C 1 -C 6 ) haloalkoxy, halogen, OH, or NH 2 , wherein the alkyl or alkoxy is optionally substituted with one or more substituents selected from NR 7 R 8 , phenyl, and 5- or 6-membered heteroaryl containing 1 or 2 heteroatoms selected from N and S; each R 6 is independently (C 1 -C 6 ) alkyl, (C 1 -C 6 ) haloalkyl, (C 1 -C 6 ) alkoxy, (C 1 -C 6 ) haloalkoxy, halogen, OH, or NH 2 , wherein the alkyl or alkoxy is optionally substituted with one or more substituents selected from NR 7 R 8 , phenyl, and 5- or 6-membered heteroaryl containing 1 or 2 heteroatoms selected from N and S; each R 7 and R 8 is independently H, (C 1 -C 4 ) alkyl, or (C 1 -C 4 ) haloalkyl; and t is 0, 1, or 2, wherein the Targeting Ligand is bonded to the Linker via attachment to A or G; wherein the Linker is of Formula LO:or an enantiomer, diastereomer, or stereoisomer thereof, wherein pl is an integer selected from 0 to 12; p2 is an integer selected from 0 to 12; p3 is an integer selected from 1 to 6, or 0 when Q is Q 1 -(O) 0-1 -Q 2 ; each W is independently absent, CH 2 , O, S, NH, or NR 19 ;Z1 is absent, OCH 2 C(O)NH, OCH 2 C(O)NR 19, C(O)NH, C(O)NR 19 , NHC(O), NR 19 C(O), C(O), CH 2 , O, NH, or NR 19 ; each R 19 is independently C 1 -C 3 alkyl; andQ is absent, NHC(O)CH 2 , O(CH 2 ) 0-2 , or Q 1 -(O) 0-1 -Q 2 ;Q1 and Q 2 are each independently absent, C 1 -C 4 alkyl enyl, cyclopropyl, cyclobutyl, cyclopen- tyl, phenyl, pyridinyl, pyrimidinyl, pyrazinyl, or 5- or 6-membered heterocyclyl containing 1 or 2 N, wherein the Linker is covalently bonded to the Degron via thenext to Q, and covalently bonded to the Targeting Ligand via the next to Z 1:wherein the Degron is of Formula D1, D2, or D3 :or an enantiomer, diastereomer, or stereoisomer thereof, wherein:Y1 is a bond, (CH 2)1-6, (CH 2 ) 0-6 — O, (CH 2 ) 0-6 — C(O)NR 11 , (CH 2 ) 0-6 —NR 11 C(O), (CH 2 ) 0-6 — NH, or(CH2)0-6— NR 12 ;Z is C(O) or C(R 13 ) 2 ;R11 isHorC 1 -C 6 alkyl;R12 is C 1 -C 6 alkyl or C(O)— C 1 -C 6 alkyl;each R 13 is independently H or C 1 -C 3 alkyl; each R 14 is independently C 1 -C 3 alkyl;R15 is H, deuterium, C 1 -C 3 alkyl, F, or Cl; each R 16 is independently halogen, OH, C 1 -C 6 alkyl, or C 1 -C 6 alkoxy; q is O, 1, or 2; v is O, 1, 2, or 3; each R 17 is independently C 1 -C 3 alkyl; q' is 0, 1, 2, 3 or 4;R18 is H or C 1 -C 3 alkyl;R20 is t-butyl or i-propyl;R21 isand bonded to the carbon atom marked with * with the nitrogen atom, or 5- or 6-membered het- eroaryl containing 1 or 2 heteroatoms selected from N, O, and S; andR22 is H or C 1 -C 3 alkyl, wherein the Degron is covalently bonded to the Linker via14. The compound for use according to any one of claims 1 to 11, wherein said compound capable of inhibiting TRIM24 activity is selected from the following compounds:15 A pharmaceutical composition comprising a compound capable of inhibiting TRIM24 activity and an androgen deprivation drug for use in a method of treatment of a hormone- sensitive meta-static prostate cancer, optionally in combination with radiotherapy.16 A pharmaceutical composition comprising a compound capable of inhibiting TRIM24 activity and one or more excipients for use in a method of treatment of a castration-resistant prostate cancer, preferably for use in a method of treatment of castration-resistant prostate can- cer driven by AR-V7 or other AR isoforms lacking the ligand-binding domain.17 A pharmaceutical composition comprising a compound capable of inhibiting TRIM24 activity and one or more excipients for use in a method of treatment of a patient with a castra- tion-resistant prostate cancer, wherein the cancer of said patient is positive for the expression of AR-V7 or other AR isoforms lacking the ligand-binding domain.

18. A pharmaceutical composition for use according to any one of the claims 15 to 17, wherein said compound capable of inhibiting TRIM24 activity is the compound defined in any one of the claims from 12 to 14.

19. A pharmaceutical composition for use according to claim 15, wherein said androgendeprivation drug is leuprorelin (leuprolide), goserelin, triptorelin, histrelin, buserelin and / or de- garelix.