Lineage perturbation therapy methods including LATS1 / 2 kinase inhibitors and tip60 inhibitors

Abstract

Disclosed herein are methods including LATS kinase inhibitors or TIP60 inhibitors to perturb lineages within heterogenous tumors (e.g., prostate cancer) and enhance responsiveness to Androgen Receptor Signaling Inhibitor (ARSI) therapy or other therapeutic agents that target adenocarcinoma lineages (e.g., PSMA or STEAP1-expressing tumor cells.

Description

Atty. Dkt. No. 115872-3363LINEAGE PERTURBATION THERAPY METHODS INCLUDING LATS1 / 2 KINASE INHIBITORS AND TIP60 INHIBITORSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 714,735, filed October 31, 2024, the entire contents of which are incorporated herein by reference.STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with government support under CA092629, CA265768, and CA155169 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] Provided herein are methods of using LATS kinase inhibitors or TIP60 inhibitors to perturb lineages within heterogenous tumors (e.g., prostate cancer) and enhance responsiveness to Androgen Receptor Signaling Inhibitor (ARSI) or other therapeutic agents that target adenocarcinoma lineages (e.g., PSMA or STEAP1 -expressing tumor cells).BACKGROUND

[0004] Lineage plasticity — the ability of cancer cells to adopt alternative identities in response to environmental or therapeutic pressures — is increasingly recognized as a major mechanism of resistance to targeted therapies across multiple cancer types, including prostate cancer. For example, the mainstay of prostate cancer treatment is androgen deprivation therapy (ADT), including next-generation AR pathway inhibitors (ARPIs) such as enzalutamide and abiraterone. Despite improved overall survival, an unintended consequence of the selective pressure of ARPI therapy is an increase in treatment-related neuroendocrine prostate cancer (NEPC), a lineage transition from prostate adenocarcinoma (PRAD). Once this lineage switch occurs, NEPC is associated with poor clinical outcomes and is largely unresponsive to existing treatments. Accordingly, there is an urgent need for new therapeutic methods and compositions that perturb lineages within heterogenous tumors (e.g., prostate cancer) and enhance responsiveness to Androgen Receptor Signaling Inhibitor (ARSI) or other therapeutic agents that target adenocarcinoma lineages (e.g., PSMA or STEAP1 -expressing tumor cells).14932-3629-0421 .1Atty. Dkt. No. 115872-3363SUMMARY OF THE PRESENT TECHNOLOGY

[0005] In one aspect, the present disclosure provides a method for treating or preventing prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a LATS kinase inhibitor. In another aspect, the present disclosure provides a method for enhancing responsiveness of a prostate cancer patient or a lung cancer patient to Androgen Receptor Signaling Inhibitor (ARSI) comprising sequentially, simultaneously or separately administering to the patient an effective amount of a LATS kinase inhibitor and an effective amount of an ARSI. In some embodiments, the ARSI is selected from the group consisting of abiraterone acetate, apalutamide, bicalutamide, clascoterone, enzalutamide, flutamide, ARN-509, nilutamide and darolutamide.

[0006] In yet another aspect, the present disclosure provides a method for treating prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a LATS kinase inhibitor and an effective amount of an agent that specifically targets prostate adenocarcinoma (PRAD) or lung adenocarcinoma (LU D) lineage cells in tumors. In some embodiments, the agent that specifically targets PRAD or LU AD lineage cells binds to STEAP1 or PSMA. Additionally or alternatively, in certain embodiments, the agent that specifically targets PRAD or LU AD lineage cells is an antibody drug conjugate, a T cell engager, a CAR T cell or a radioligand. In some embodiments, the radioligand is lutetium- 177-PSMA-617.

[0007] In any of the preceding embodiments of the methods described herein, the LATS kinase is LATS1 or LATS2. In some embodiments, the LATS kinase inhibitor is selected from among: TDI-011536, VT02956, GA-017, NIBR, 4-(4-(4-(4-Chlorophenyl)piperidin- 4-yl)phenyl)-lH-pyrrolo[2,3-b]pyridine, 4-(4-(Piperidin-4-yl)phenyl)-lH-pyrrolo[2,3- b]pyridine, 3-Fluoro-4-(4-(piperidin-4-yl)phenyl)-lH-pyrrolo[2,3-b]pyridine, 4-(4-(3- Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)piperidin-4-ol, 3 -(4-(3 -Fluoro- 1H- pyrrolo[2,3-b]pyridin-4-yl)phenyl)piperidin-3-ol, 3-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin- 4-yl)phenyl)pyrrolidin-3-ol, 3-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)azetidin- 3-ol, 4-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)azepan-4-ol, (lR,5S)-3-(4-(3- Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol, 6-(4-(3- Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-2-azaspiro[3.3]heptan-6-ol, 4-(4-(3-Fluoro- lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3-methylpiperidin-4-ol, (3R,4s,5S)-4-(4-(3-Fluoro- lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)-4-(2-Fluoro-24932-3629-0421 .1Atty. Dkt. No. 115872-33634-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)- 4-(2-Chloro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)-4-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-2-methylphenyl)-3,5- dimethylpiperidin-4-ol, (3S,4s,5R)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)- 6-methylphenyl)-3,5-dimethylpiperidin-4-ol, (3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH- pyrrolo[2,3-b]pyridin-4-yl)-6-methylphenyl)-l,3,5-trimethylpiperidin-4-ol, (3R,4s,5S)-4-(2- Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-6-methylphenyl)-l-isopropyl-3,5- dimethylpiperidin-4-ol, (3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)- 6-methylphenyl)-3,5-dimethyl-l-(oxetan-3-yl)piperidin-4-ol, l-((3R,4s,5S)-4-(2-Fluoro-4- (3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-6-methylphenyl)-4-hydroxy-3,5- dimethylpiperidin-l-yl)ethan-l-one, 4-((3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3- b]pyridin-4-yl)-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-l-yl)tetrahydro-2H- thiopyran 1,1 -di oxide,34932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0008] In one aspect, the present disclosure provides a method for treating or preventing prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a TIP60 inhibitor. In another aspect, the present disclosure provides a method for enhancing responsiveness of a prostate cancer patient or a lung cancer patient to Androgen Receptor Signaling Inhibitor (ARSI) comprising sequentially, simultaneously or separately administering to the patient an effective amount of a TIP60 inhibitor and an effective amount of an ARSI. In some embodiments, the ARSI is selected from the group consisting of abiraterone acetate, apalutamide, bicalutamide, clascoterone, enzalutamide, flutamide, ARN-509, nilutamide and darolutamide.

[0009] In yet another aspect, the present disclosure provides a method for treating prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a TIP60 inhibitor and an effective amount of an agent that specifically targets prostate adenocarcinoma (PRAD) lineage cells or lung adenocarcinoma (LU AD) lineage cells in tumors. In some embodiments, the agent that specifically targets PRAD or LU AD lineage cells binds to STEAP1 or PSMA. Additionally or alternatively, in certain embodiments, the agent that specifically targets PRAD or LU AD lineage cells is an antibody drug conjugate, a T cell engager, a CAR T cell or a radioligand. In some embodiments, the radioligand is lutetium- 177-PSMA-617. In some embodiments, the TIP60 inhibitor is selected from among TH1834, NU9056, Pentamidine, Acetyl CoA, and MG149. In some embodiments, the TIP60 inhibitor is selected from among:44932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0010] Additionally or alternatively, in some embodiments, the ARSI is administered orally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, subcutaneously, intratumorally, topically, by inhalation spray, buccally, or via an implanted reservoir. Examples of ARSIs include, but are not limited to abiraterone acetate, apalutamide, bicalutamide, clascoterone, enzalutamide, flutamide, ARN-509, nilutamide and darolutamide.

[0011] In any and all embodiments of the methods disclosed herein, the prostate cancer or lung cancer harbors a mutation in Rbl and / or Trp53. Additionally or alternatively, in some embodiments, the prostate cancer or lung cancer comprise ASCL1+ cells.Additionally or alternatively, in certain embodiments, the TTP60 inhibitor or the LATS1 / 2 inhibitor is administered orally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, subcutaneously, intratumorally, topically, by inhalation spray, buccally, or via an implanted reservoir.

[0012] In any and all embodiments of the methods disclosed herein, the prostate cancer is neuroendocrine prostate cancer (NEPC) or the lung cancer is lung neuroendocrine cancer.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGs. 1A-1J show the removal of ECM activates Ascii expression. FIG. 1A: IHC of Vimentin and Fibronectin, along with Masson’s tri chrome staining of normal prostate tissue, adenocarcinoma regions, and neuroendocrine regions from TKO tumors. FIG. IB: Deconvolved NEPC theta estimates and corresponding stromal theta estimates across two specimens. Spatial maps highlight the distribution of NEPC and fibroblast-like content across Visium spots. FIG. 1C: Proportion of spots classified as both tumor-high54932-3629-0421 .1Atty. Dkt. No. 115872-3363 and stroma-high for PRAD compared to NEPC. FIG. ID: ASCL1 IHC of TKOM organoids cultured in Matrigel versus suspension conditions. FIG. IE: Volcano plot showing differentially expressed genes from RNA-seq analysis comparing TKOM organoids cultured in suspension versus Matrigel. FIG. IF: Gene set enrichment analysis (GSEA) of RNA-seq data from TKOM organoids in suspension versus Matrigel culture. FIG. 1G: UMAP of scRNA-seq data from TKOM organoids under the indicated conditions. FIGs. 1H-1J: UMAPs showing Ascii expression (FIG. 1H), Ascii peaks (FIG. II) and Foxa2 expression (FIG. 1 J) in TKOM organoids, corresponding to the conditions in FIG. 1G.

[0014] FIGs. 2A-2I show that the loss of ECM-integrin signaling promotes PRAD to NEPC lineage transition. FIG. 2A: ASCL1 IHC of TKOM organoids 17 days after electroporation with control sgRNA (sgNT) or sgRNA targeting Itgbl (sgltgbl). FIG. 2B: Western blot confirming Itgbl knockout in TKOM organoids. FIG. 2C: Quantitative reverse-transcription PCR (qRT-PCR) analysis showing expression of NE markers at day 8, 12 and 17 following Itgbl deletion. Error bars represent ±SEM, n=3. FIG. 2D: Heatmap showing differential expression of integrin genes in TKOM organoids cultured in Matrigel versus suspension. FIG. 2E: Schematic diagram of the replating experiment. FIG. 2F: qRT-PCR of neuroendocrine markers in TKOM organoids cultured in Matrigel, suspension conditions and replating back to Matrigel. Error bars represent ±SEM, n=3. FIG. 2G: qRT- PCR showing the expression of multiple integrin genes in TKOM organoids cultured in Matrigel, suspension conditions and after replating back in Matrigel. Error bars represent ±SEM, n=3. FIG. 2H: scRNA-seq analysis of PtRP (TKO) GEMMs showing reduced integrin gene expression score in NEPC compared to PRAD. FIG. 21: Box plot demonstrating reduced integrin gene expression score in NEPC (CRPC-NE) compared to PRAD (CRPC-adeno) in two published patient cohorts. The integrin gene expression score represents the mean expression of all integrin genes.

[0015] FIGs. 3A-3K show that YAP1 / TEAD activation blocks Ascii induction through ECM / integrin engagement. FIGs. 3A-3B: Western blot analysis of representative kinases, substrates and target genes downstream of integrin downstream in TKOM organoids in Matrigel or after one day in suspension culture (FIG. 3A) or two days after Itgbl knockout (FIG. 3B). FIG. 3C: qRT-PCR for Ascii in TKOM organoids after treatment with pharmacologic inhibitors of the indicated targets. FAKi: 5uM PF573228; SFKi: 5uM Dasatinib; PI3Ki: 2uM GDC0941; MEKi: luM AZD6244; TEADi: luM IAG933. Error bars, ±SEM, n=3. FIG. 3D: qRT-PCR of the YAP1 / TAZ target genes Ctgf and Cyr61 in64932-3629-0421 .1Atty. Dkt. No. 115872-3363TKOM organoids after treating with the indicated inhibitors. Error bars, ±SEM, n=3. FIG. 3E: qRT-PCR of NE genes in RPWEa / ?7^? ' / r^rand TK0M / Ea / ?7^ ' / r^rorganoids 6 days and 10 days following Adeno-Cre (Ad-Cre) infection. Error bars, ±SEM, n=3. FIG. 3F: qRT-PCR of NE markers in RPM and TKOM organoids overexpressing EV, constitutive YAP5SA or TAZ4SA, in cultured in Matrigel or in suspension for 14 days. Error bars, ±SEM, n=3. FIGs. 3G-3H: Multiplex IF of phospho-FAKY397, active-YAPl and ASCL1 of tumor sections from 12-week-old TKO GEMM (FIG. 3G) with quantification (FIG. 3H). FIGs. 3I-3K: Box plot demonstrating reduced expression of YAP1 (FIG. 31) and WWTR1 (FIG. 3 J), as well as decreased YAP / TAZ target score (FIG. 3K), in NEPC (CRPC-NE) compared to PRAD (CRPC-adeno) tumors from the SU2C cohort.

[0016] FIGs. 4A-4J shows that LATS inhibition impairs acquisition and maintenance of NE lineage. FIG. 4A: Cartoon showing ECM-Integrin inhibits LATS 1 / 2 to activate YAP1 / TAZ / TEAD. FIG. 4B: Schematic diagram illustrating the 3 treatment scenarios in (FIGs. 4C-4E) FIG. 4C: Western blot analysis of indicated proteins in TKOM organoids cultured in Matrigel or suspension, treated with 5mM TRULI for 4 hours. FIG. 4D: qRT- PCR of NE genes (Ascii, Foxa2, Insml, and Chgd) in TKOM organoids cultured in Matrigel, suspension, or suspension plus 5mM TRULI for 14 days. Error bars represent ±SEM, n=3. FIG. 4E: Western blot analysis of indicated targets in RPM NE organoids following treatment with 5mM TRULI for 4 days. FIG. 4F: qRT-PCR analysis of ASCL1 and YAP1 / TAZ targets (CTGF and CYR61) in MSKPCA14 and MSKPCA24 patient- derived organoids treated with DMSO, 5uM TRULI, lOuM TRULI for 7 days. Error bars represent ±SEM, n=3. FIG. 4G: ASCL1 and YAP1 IHC in MSKPCA14 patient-derived organoids with DMSO, 5uM TRULI, or lOuM TRULI for 7 days. FIG. 4H: Quantification of the percentage of Ascii positive cells and nuclear YAP1 positive cells. Error bars represent ±SEM, n=4, ***p<0.0005, ****p<0.0001. FIG. 41: Representative H&E images and Ascii IHC images of RPM transplantation tumors overexpressing EV or wild-type YAP1 or YAP15SA. FIG. 4J: Quantification of the percentage of Ascii positive cells in FIG. 41. Error bars represent ±SEM, n=12, ****p<0.0001.

[0017] FIGs. 5A-5C shows the reconstitution of in vivo PRAD to NEPC lineage transition in vitro. FIG. 5A: ASCL1 IHC in TKO PRAD organoids with DMSO or TEADi (luM IAG933) for eight days. The scale bar represents 50mm. FIG. 5B: Principal component analysis (PC A) of RNA-seq data from TKO PRAD organoids subjected to the74932-3629-0421 .1Atty. Dkt. No. 115872-3363 indicated treatments. FIG. 5C: Western blot of NEPC and PRAD lineage markers in TKO PRAD organoids and NEPC tumoroids.

[0018] FIGs. 6A-6E show that FOXA1 is essential for the PRAD to NEPC lineage transition. FIG. 6A: CUT&RUN profiles of TEAD1, YAP1 and FOXA1 in TKO PRAD and NEPC organoids. Clusters were defined based on differential TEAD1 binding between PRAD and NEPC. FIGs. 6B-6C: Pathway enrichment analysis of TEAD1 CUT&RUN peaks in PRAD (FIG. 6B) and NEPC (FIG. 6C). Pathways are ranked by gene ratio. FIG. 6D: Genome browser tracks showing CUT&RUN profiles of YAP 1 and TEAD1 binding at the adenocarcinoma marker Tacstd2 (TROP2) and the NE gene Ascii in TKO PRAD and NEPC organoids. Screenshots were generated using Integrative Genomics Viewer (IGV). FIG. 6E: Top enriched transcription factor binding motifs identified by HOMER known- motif analysis of TEAD1 CUT&RUN peaks in TKO PRAD and NEPC organoids.

[0019] FIG. 7 shows a model of how integrin-mediated YAP activation limits TEAD and FOXAl-driven PRAD to NEPC lineage transition. In PRAD, ECM-integrin signaling inhibits LATS1 / 2 to activate YAP / TEAD, which cooperate with AR and FOXA1 to maintain PRAD lineage identity. Combined inhibition of YAP / TEAD and AR pathways fully reprograms PRAD cells into NEPC lineage. During this transition, TEAD and FOXA1 relocalize from PRAD cis-regulatory elements (CREs) to NEPC CREs, driving ASCL1 induction and commitment to the neuroendocrine lineage.

[0020] FIGs. 8A-8D show that cancer-associated fibroblasts and ECM are depleted in NEPC. FIG. 8A: Western blot analysis confirming disruption of Trp53, Rbl and Pten, as well as overexpression of cMYC in engineered mutant organoid lines. FIG. 8B: Hematoxylin and eosin staining (H&E) and Ascii immunohistochemistry (IHC) of TKO, RPM and TKOM organoids, as well as their corresponding orthotopic transplant tumors. FIG. 8C: Vimentin IHC and Masson’s trichrome staining of early- and late-stage orthotopic transplants derived from TKO and RPM organoids. FIG. 8D: Deconvolved NEPC theta estimates and corresponding stromal theta estimates across three specimens. Spatial maps highlight the distribution of NEPC and fibroblast-like content across Visium spots.

[0021] FIGs. 9A-9C show that extracellular matrix removal induces Ascii expression. FIG. 9A: Time-course qRT-PCR analysis showing progressive induction of Ascii in TKOM organoids cultured under suspension conditions. Error bars represent ±SEM, n=3. FIG. 9B: Heatmap displaying expression levels of NEPC transcription factors in TKOM84932-3629-0421 .1Atty. Dkt. No. 115872-3363 organoids cultured in Matrigel versus suspension. FIG. 9C: UMAP from scRNA-seq showing expression of Insml and Foxal in TKOM organoids corresponding to the conditions in (FIG. 1G)

[0022] FIGs. 10A-10C show that YAP1 / TAZ / TEAD suppresses Ascii induction and PRAD-to-NEPC transition. FIG. 10A: qRT-PCR analysis of YAP1 / TAZ target genes in RPM and TKOM organoids at 6 and 10 days following Yapl and Wwtrl deletion via Adeno-Cre (Ad-Cre) infection. Error bars represent ±SEM, n=3. FIG. 10B: qRT-PCR analysis of integrin gene expression in RPM and TKOM organoids at 6 and 10 days following Yapl and Wwtrl deletion via Ad-Cre. Error bars represent ±SEM, n=3. FIG. 10C: qRT-PCR analysis of YAP1 / TAZ target genes in RPM and TKOM organoids culturing in Matrigel, or in suspension for 14 days while overexpressing either empty vector (EV), constitutively active YAP (YAP5SA) or constitutively active TAZ (TAZ4SA). Error bars represent ±SEM, n=3.

[0023] FIGs. 11A-11F show that LATS inhibition impairs acquisition and maintenance of neuroendocrine state. FIG. 11A: qRT-PCR of YAP / TAZ target genes (Cyr61, Ptpnl4 andAxT) in TKOM organoids cultured in Matrigel, suspension, or suspension plus 5mM TRULI for 14 days. Error bars represent ±SEM, n=3. FIG. 11B: UMAP of epithelial cells in TKO GEMM tumors by scRNA-seq, showing that Itgav is silenced in NEPC cells but highly expressed in adenocarcinoma cells. NEPC cells are positive for Chga, whereas adenocarcinoma cells are Chga-negative. FIG. 11C: Schematic illustration of the workflow used to generate NEPC tumoroids from PRAD organoids. FIG. 11D: Representative FACS plots showing sorting of Epcam / Integrinav" cells from TKO or RPM tumors. FIG. HE: Representative Ascii H4C images of organoids derived by sorting Epcam / Integrinav" cells from TKO or RPM orthotopic tumors. The scale bar represents 100mm. FIG. HF: Western blot analysis of phospho-Yapl (S127) in mouse prostates following treatment with vehicle or TDI-011536 for 2 or 4 hours.

[0024] FIG. 12 shows normalized counts of indicated neuroendocrine markers in TKO PRAD organoids treated with DMSO or TEADi, and in TKO NEPC tumoroids. Error bars, ±SEM; n=3.

[0025] FIGs. 13A-13C show that TEAD and FOXA1 are implicated in the PRAD to NEPC lineage transition. FIG. 13A: qRT-PCR showing decreased expression of NEPC markers following Teadl knockout in TKO NEPC organoids. Error bars represent ±SEM,94932-3629-0421 .1Atty. Dkt. No. 115872-3363 n=3, ****p<0.0001. FIG. 13B: Western blot analysis demonstrating reduced protein level of NE markers upon Teadl knockout in TKO NEPC organoids. FIG. 13C: Western blot analysis showing partial knockout of Tead proteins by CRISPR-Cas9 RNP in PRAD organoids.

[0026] FIGs. 14A-14L show that isogenic organoid models reveal epigenetic reprogramming during prostate cancer neuroendocrine transformation. FIG. 14A: Schematic showing the derivation of isogenic PRAD and NEPC organoids using in vivo neuroendocrine transformation. FIG. 14B: Hematoxylin and eosin (H&E) and multiplex immunofluorescence (IF) of representative PRAD and NEPC tumors from subcutaneously transplanted TKO organoids. VIM (Vimentin), ECAD (E-cadherin), SYP (Synaptophysin). Scale bar representing 50 pm applies to all panels. FIG. 14C: H&E and immunofluorescence (IF) of representative WT, PRAD and NEPC organoids. GFP indicates Cre-induced recombination (TKO). Scale bars representing 10 pm apply to all H&E and IF panels, respectively. FIG. 14D: Flow cytometric analysis of DLL3 expression in PRAD and NEPC organoids. FIG. 14E: Principal component analysis (PCA) of RNA-seq using WT, PRAD and NEPC organoids. FIG. 14F: Heatmap of normalized enrichment score (NES) from GSEA analysis of RNA-seq data showing significantly enriched gene sets in indicated PRAD and NEPC organoid lines (FDR < 0.05). Differentially expressed genes were identified by comparing individual PARE) and NEPC line with both lines of the other lineage. Custom expression signatures of genes enriched in WT epithelial cells (luminal 1, luminal2, basal) of intact mouse prostate, genes enriched in PRAD or NEPC tumors from the TKO GEMM model, genes upregulated in NEPC (vs. PRAD) patient tumor samples in two clinical cohorts (SU2C and WCM), other neuroendocrine / neuronal signatures, and hallmark inflammatory signaling signatures were analyzed. FIG. 14G: Heatmap showing z- score transformed expression of luminal, basal and neuroendocrine marker genes from RNA-seq of indicated organoid lines. FIG. 14H: Longitudinal tumor volumes after subcutaneous transplant of indicated organoids. FIG. 141: PCA of ATAC-seq at all peaks across WT, PRAD and NEPC organoids. FIG. 14J: Heatmap showing the emission parameters of the 9-state chromatin model built using chromoHMM with binarized CUT&RUN data from six histone modifications. The darker blue color indicates higher probability of observing the modification in the state. FIG. 14K: Heatmap showing proportion of overlap (in bases) of chromatin states between tumor (PRAD and NEPC) and WT organoids across the genome as quantification of chromatin state changes. FIG. 14L:104932-3629-0421 .1Atty. Dkt. No. 115872-3363Left, Venn diagram showing overlap of non-TSS H3K27ac peaks, which are used to define active enhancers, in PRAD (AD2) and NEPC (NE2). Right, heatmap showing enrichment of enhancer features (H3K27ac, H3K4mel, accessibility) at NEPC-specific, PRAD-specific and shared active enhancers across WT, PRAD and NEPC organoid lines.

[0027] FIGs. 15A-15G show the identification of lineage survival transcription factor families in NEPC. FIG. 15A: (Top) Venn diagram showing overlap of ATAC-seq peaks in PRAD (ADI and AD2) and NEPC (NE1 and NE2). CRE, c / .s-regulatory elements. (Bottom) Top 5 de novo TF motifs enriched at NEPC-specific ATAC-seq peaks (target coverage > 10%, ranked by p values). FIG. 15B: Volcano plot showing differential RNA expression of TFs in NEPC (NE2) vs. PRAD (AD2). Screen candidates were highlighted in red and blue. FIG. 15C: Schematic showing cell competition assay to assess cell proliferation upon knockout of individual genes. Percentage of BFP+ sgRNA-containing cells were monitored over a time course of 4 passages (PO-4). BFP% at day 2 (P0) was used as a baseline representing transduction efficiency. FIG. 15D: Cell competition assays using at least two independent sgRNAs targeting the DNA binding domains of 25 individual TFs identified in FIG. 15A and FIG. 15B in indicated organoid lines, showing relative BFP percentages at P4 (day 18 for NE2 and AD2 and day 31 for NE1) after sgRNA transduction (see supplementary data for complete set of measurements). BFP percentages were normalized to day 2 (P0) and the NegCtrl4 at the respective day (relative %BFP+). The five families of NEPC lineage survival TFs were highlighted. sgRNAs targeting common essential genes implicated in DNA replication (Mcm2 and Pend) were used as positive controls. Dashed lines represent 100%. Each dot represents a sgRNA and is the average of two technical replicates. Data represent mean ± SD from n = 2 to 4 biological replicates (independent sgRNAs). FIG. 15E: Cell competition assays using two independent single or dual sgRNA vectors targeting either or both TF paralogs in indicated organoid lines, showing relative BFP percentage over 4 passages. BFP percentages were normalized to day 2 (P0) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIG. 15F: mRNA expression (bulk RNA-seq) of key members of the five NEPC lineage survival TF families in histologically validated PRAD and NEPC tumor samples of two patient cohorts (SU2C and WCM). SU2C, n = 210 for PRAD and n = 22 for NEPC. WCM, n = 34 for PRAD and n = 15 for NEPC. FIG. 15G: Cell competition assays using two independent sgRNAs targeting the DNA binding domains of key members of the five families of NEPC lineage survival TFs in indicated NEPC PDO lines, showing relative114932-3629-0421 .1Atty. Dkt. No. 115872-3363BFP percentages at P4 (day 31) after sgRNA transduction (see supplementary data for complete set of measurements). BFP percentages were normalized to day 3 (PO) and the NegCtrl4 at the respective day (relative %BFP+). sgRNAs targeting Mcm2 and Pena were used as positive controls. Dashed lines represent 100%. Each dot represents a sgRNA and is the average of two technical replicates. Data represent mean ± SD from n = 2 biological replicates (independent sgRNAs).

[0028] FIGs. 16A-16G show the identification of TIP60 as a critical lineage-biased dependency in NEPC. FIG. 16A: Cell competition assays using at least two independent single or dual sgRNA vectors targeting the catalytic domains of either or both paralogs of chromatin regulators in indicated organoid lines, showing relative BFP percentage over 4 passages (Pl -4) after sgRNA transduction. BFP percentages were normalized to PO (day 2) and the NegCtrl4 at the respective day (relative %BFP+). Subunits of the TIP60 complex (Tip60 and Ep400) were highlighted. Dashed lines represent 100%. Data represent mean ± SD from n = 2 technical replicates. FIG. 16B: Cell competition assays using two independent TIP60 sgRNAs in indicated PRAD and NEPC PDOs, showing relative BFP percentage over 4 passages (Pl -4). BFP percentage was measured only on P0, P2 and P4 for LuCaP49. BFP percentages were normalized to day 3 (P0) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIG. 16C: Longitudinal tumor volumes after subcutaneous transplant of NE2 organoids 3 days after transduction with NegCtrl or Tip60 sgRNAs. sgRNA-containing cells were sorted out based on BFP positivity. Data represent mean ± SEM from n = 5 tumors for sgNegCtrl_4 and n = 4 tumors for sgTip60_l and 4. FIG. 16D: Tumors harvested on day 31 after transplant of NE2 organoids transduced with indicated sgRNAs. Percentages of BFP+ sgRNA-containing cells in the GFP+ tumor cell populations using flow cytometric analysis of dissociated tumors were labeled. FIG. 16E: Schematic showing the strategy for chemical genetic targeted degradation of TIP60. dTAG-Tip60 lines were engineered by lentiviral expression of a dTAG-Tip60 transgene and knockout of endogenous Tip60. Targeted proteolysis was induced by treatment of bifunctional degraders dTAGv-l or dTAG-13 for proteasome dependent degradation. FIG. 16F: Western blots of whole cell extract from indicated dTAG-Tip60 lines treated with a 3-fold gradient of dTAGv-l for 6 hours. dTr4 indicates Tip60 knockout with sgTip60_4 and rescue expression of dTAG-Tip60. Actin was used as loading control. FIG. 16G: dTAGv-l dose response curves and IC50 values for indicated dTAG-Tip60 and parental lines (no dTAG-Tip60 or Tip60 sgRNA). Cell lines124932-3629-0421 .1Atty. Dkt. No. 115872-3363 were treated with the same dTAGv-l gradient as in FIG. 16F for 6 days before CellTiter Gio assays were performed to determine cell viability. Data represent mean ± SD from n = 10 biological replicates.

[0029] FIGs. 17A-17O shows that TIP60 acts as a coactivator of MYCL to integrate pro-growth transcriptional outputs of key NEPC TFs and promote lineage survival. FIG. 17A: Heatmap showing occupancy (CUT&RUN) of indicated histone modifications and proteins and chromatin accessibility (ATAC-seq) at NEPC, PRAD and shared CREs defined by ATAC-seq in NE2 and AD2 organoids. FIG. 17B: (Top) Venn diagram depicting overlap of CUT&RUN peaks of indicated TFs with TIP60 peaks. (Bottom) Bar graph showing the percentages of CUT&RUN peaks of indicated TFs that overlap with TIP60 peaks. FIG. 17C: PCA of RNA-seq of NE2 organoids upon 3-day knockout of indicated gene(s). sgRNA-containing cells were sorted out based on BFP positivity 3 days after transduction of sgRNA vectors. Double knockout of Foxal / 2 and Soxl / 11 were performed, n = 3 independent sgRNA transductions for sgCtrl3, sgTip60, sgMycl and sgNfib. n = 4 independent sgRNA transductions for sgAscll, sgSoxl / 11, sgFoxal / 2. FIG. 17D: Overrepresentation analyses (FDR < 0.05) of custom neuroendocrine / neuronal signatures and hallmark gene sets in downregulated genes upon 3-day knockout of indicated genes compared to control sgRNA in NE2 organoids. FIG. 17E: Western blot analyses of immunoprecipitation using FLAG tag in NE2 organoids expressing lentiviral HA-FLAG- tagged Tip60 or empty vector (EV). TIP60c, the TIP60 complex. FIG. 17F: Western blots of NE2 Mycl dTrl organoids treated with dTAG-13 or dTAGv-l for 24 h. Mycl dTrl indicates Mycl knockout with sgMycl_l and rescue expression of dTAG-Mycl (with 2xHA and 3xFLAG tags). FIG. 17G: Cell viability of NE2 Mycl dTrl organoids after 6 days of treatment of 500 nM dTAG-13 or dTAGv-l. Data represent mean ± SD from n = 10 biological replicates. FIG. 17H: Average profile showing occupancy (CUT&RUN) of MYCL (via HA tag) and TIP60 at TIP60 peaks in NE2 Mycl dTrl organoids treated with 500 nM dTAGv-l for 6 h. NT, non-treated. FIG. 171: Differential expression (RNA-seq) of indicated genes upon 3-day knockout of indicated genes compared to control sgRNA in NE2 organoids. FIG. 17J: Genome browser view of chromatin accessibility (ATAC-seq) and occupancy (CUT&RUN) of indicated TFs at the Mycl locus in AD2 or NE2 organoids. FIG. 17K: Cell competition assays using two independent single or dual sgRNA vectors targeting indicated TF or TF paralogs in parental NE2 organoids or NE2 overexpressing Mycl, showing relative BFP percentage over 4 passages. BFP percentages were normalized134932-3629-0421 .1Atty. Dkt. No. 115872-3363 to day 2 (PO) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIG. 17L: Boxplot of single-sample GSEA (ssGSEA) scores (RNA-seq) showing expression of hallmark MYC target genes in PRAD and NEPC patient tumor samples of the SU2C cohort. FIG. 17M: Cell competition assays using two independent Mycl sgRNAs in parental NE2 organoids or NE2 overexpressing Myc, Mycn, or Mycl, showing relative BFP percentage over 4 passages. BFP percentages were normalized to day 2 (PO) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIGs. 17N and 170: (Left) Western blots of whole cell extract from NE2 organoids with ectopic expression of dTAG- Myc (FIG. 17N) or dTAG-Mycn (FIG. 170) and knockout of Mycl using sgMycl_2 (Myc dTrl2 and Mycn dTrl2), which were treated with 500 nM dTAG-13 or dTAGv-l for 24 h. (Right) Average profile showing occupancy (CUT&RUN) of TIP60 in NE2 Myc dTrl2 (FIG. 17N) or Mycn dTrl2 (FIG. 170) organoids treated with 500 nM dTAGv-l for 6 h. NT, non-treated.

[0030] FIGs. 18A-18R show the acetyltransferase and chromatin reader activities of the TIP60 complex are required for NEPC survival. FIG. 18A: Schematic showing the TIP60 complex. Red square indicates acetyltransferase domain. Red outlines indicate subunits with chromatin reader domain(s). Black circles indicate histone modifications reportedly / typically recognized by respective reader subunits. FIG. 18B: Western blots of whole cell extract from NE2 organoids overexpressing wild type (WT) or Q377E / G380E catalytically dead (CD) TIP60. In FIGs. 18B, 18D, 18E, 18H, 181, 18N, and 180, Actin was used as loading control. FIG. 18C: Cell competition assays using two independent Tip60 sgRNAs in parental NE2 organoids or NE2 overexpressing WT or CD TIP60, showing relative BFP percentage over 4 passages. BFP percentages were normalized to day 2 (PO) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIG. 18D: Western blots of whole cell extract from NE2 organoids with Tip60 knockout. sgRNA-containing cells were sorted out based on BFP positivity 3 days after transduction. FIG. 18E: Western blots of whole cell extract from NE2 dTAG-Tip60 (dTr4) organoids treated with 500 nM dTAGv-l for indicated time course. FIG. 18F: Heatmap showing occupancy (CUT&RUN) of indicated proteins and histone modifications at all TIP60 peaks (promoter and distal) in NE2 organoids. FIG.18G: Average profile showing occupancy (CUT&RUN) of TIP60, H2A.Z and H2A.Zac at TIP60 peaks in NE2 dTAG-Tip60 organoids (dTr4) treated with 500 nM dTAGv-l for 4 h.144932-3629-0421 .1Atty. Dkt. No. 115872-3363NT, non-treated. FIG. 18H: Western blots of whole cell extract from NE2 organoids with Ep400 knockout. In FIGs. 18H, 181, 18N, and 180, sgRNA-containing cells were selected by puromycin treatment. FIG. 181: Western blots of whole cell extract from NE2 organoids with Srcap knockout. FIG. 18J: Cell competition assays using two independent single or dual sgRNA vectors targeting either or both H2A.Z isoforms in NE2 organoids, showing relative BFP percentage over 4 passages. BFP percentages were normalized to day 2 (PO) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIG. 18K: Western blots of whole cell extract from NE2 organoids expressing HA-tagged canonical H2A or WT / mutant H2A.Z1. 2KR, K4 / 7R mutant. 5KR, K4 / 7 / 11 / 13 / 15R mutant. H3 was used as loading control. FIG. 18L: Cell competition assays using two independent H2azl sgRNAs in NE2 organoids overexpressing an H2A / H2A.Z series, showing relative BFP percentage over 4 passages. BFP percentages were normalized to day 2 (PO) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIG. 18M: Cell competition assays using two independent single or dual sgRNA vectors targeting indicated individual TIP60 complex subunits or both paralogs in NE2 organoids, showing relative BFP percentage over 4 passages. Reader domain targeting sgRNAs were utilized for Ing3, Brd8, Yeats4, Mbtdl, Mrgl5 and Mrgx. BFP percentages were normalized to day 2 (PO) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIGs. 18N-18O: Western blots of whole cell extract from NE2 organoids with Brd8 (FIG. 18N) or Yeats4 (FIG. 180) knockout. FIGs. 18P-18Q: Average profile showing occupancy (CUT&RUN) of TIP60 in NE2 organoids with Brd8 (FIG. 18P) or Yeats4 (FIG. 18Q) knockout. NT, non-treated. FIG. 18R: schematics depicting the model for PRAD-to-NEPC transition.

[0031] FIGs. 19A-19J shows isogenic organoid models reveal epigenetic reprogramming during prostate cancer neuroendocrine transformation (related to FIGs. 14A-14L). FIG. 19A: Representative brightfield and fluorescent microscopy images of indicated organoid lines. Scale bar representing 20 pm applies to all panels. FIG. 19B: Growth assay showing relative cell number using CellTiter Gio assay 6 days after seeding same number of cells in organoid culture conditions. Data represent mean ± SD from n = 4 biological replicates. FIG. 19C: Heatmap showing z-score transformed expression (RNA- seq) of TKO GEMM PRAD and NEPC signature genes in indicated organoid lines. PRAD and NEPC signatures were the top 100 upregulated genes from scRNA-seq of TKO GEMM154932-3629-0421 .1Atty. Dkt. No. 115872-3363 tumors in PRAD or NEPC cells compared to all cells of other lineages / states. FIGs. 19D- 19E: GSEA enrichment plots of TKO GEMM (FIG. 19D) and patient (FIG. 19E) PRAD and NEPC signatures in indicated PRAD and NEPC organoid lines. Related to FIG. 14F. NES, normalized enrichment score. FIG. 19F: Longitudinal tumor volumes after subcutaneous transplant of indicated number of cells from NE2 organoids. FIG. 19G: Representative H&E and IHC staining of tumors from subcutaneously (SQ) and orthotopically (OT) transplanted NE2 organoids. Primary tumors (prostate) and metastases (lung / liver) were analyzed. SYP (Synaptophysin) was used as a marker for NEPC. FIG. 19H: Cell viability (CellTiter Gio assay) of indicate organoid lines treated with an AR agonist (DHT) or antagonist (Enzalutamide, ENZ) for 6 days in organoid culture medium without EGF. Data represent mean ± SD from n = 4 biological replicates. FIG. 191: Heatmaps showing the overlap enrichment of indicated chromatin states (FIG. 14J) in genomic categories, as calculated by chromHMM. FIG. 19J: Quantification of chromatin state changes comparing ADI, AD2, NE1, NE2 against WT as control. Sankey plots showing the proportion of each chromatin state (chromoHMM) in each organoid line as shown by the height of the rectangles, and the proportion of overlap (connecting ribbons) between the states in the organoid lines compared.

[0032] FIGs. 20A-20F show identification of lineage survival transcription factor families in NEPC (Related to FIGs. 15A-15G). FIG. 20A: Top 5 de novo TF motifs enriched at PRAD-specific ATAC-seq peaks (target coverage > 10%, ranked by p values). FIG. 20B: Heatmap showing z-score transformed expression (RNA-seq) of candidate TFs for CRISPR mini-screen in indicated organoid lines. FIG. 20C: Cell competition assays using at least two independent sgRNAs targeting the DNA binding domains of 25 TF candidates in indicated organoid lines, showing relative BFP percentage over 4 passages (Pl -4) after sgRNA transduction (related to FIG. 15D). BFP percentages were normalized to P0 (day 2) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIG. 20D: Western blots of whole cell extract from NE2 organoids with knockout of indicated TFs. For Ascii, Nfib and Mycl, sgRNA- containing cells were sorted out based on BFP positivity 3 days after sgRNA transduction. For Soxl, Foxal, Foxa2, Proxl and Onecut2, sgRNA-containing cells were selected by puromycin treatment. Actin was used as loading control. FIG. 20E: Boxplots showing expression (bulk RNA-seq) of indicated TFs in tumor samples of the SU2C and WCM prostate cancer patient cohorts, p values were calculated using two-tailed Wilcoxon rank164932-3629-0421 .1Atty. Dkt. No. 115872-3363 sum test. FIG. 20F: Cell competition assays using at least two independent sgRNAs targeting the DNA binding domains of indicated TFs in indicated PDO lines, showing relative BFP percentage over 4 passages (Pl -4) after sgRNA transduction (related to FIG. 15G). BFP percentage was measured only on PO, P2 and P4 for LuCaP49. BFP percentages were normalized to PO (day 2) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates.

[0033] FIGs. 21A-21J show the identification of TIP60 as a critical lineage-biased dependency in NEPC (Related to FIGs. 16A-16G). FIG. 21A: Heatmap showing z-score transformed expression (RNA-seq) of candidate chromatin coactivators for CRISPR miniscreen in indicated organoid lines. FIG. 21B: Cell competition assays using two independent TIP60 sgRNAs in indicated PRAD and NEPC PDOs, showing relative BFP percentage over 4 passages (Pl -4) (related to FIG. 16B). BFP percentage was measured only on PO, P2 and P4 for LuCaP49. BFP percentages were normalized to day 3 (PO) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIG. 21C: Flow cytometric analysis of dissociated tumor showing the percentage of BFP+ sgRNA-containing cells among all GFP+ tumor cells (related to FIG. 16D). Data represent mean ± SD from n = 5 (sgNegCtrl_4) and n = 4 (sgTip60_l and 4) tumors. FIG. 21D: Western blots of whole cell extract of indicated cell / organoid lines stably overexpressing dTAG-Tip60 and treated with dTAG-13 or dTAGv-l for 24 h. Actin was used as loading control. FIG. 21E: Cell competition assays using two independent Tip60 sgRNAs in indicated cell / organoid lines stably overexpressing dTAG-Tip60 and nontreated (NT) or treated with dTAG-13 or dTAGv-l for the duration of the experiment, showing relative BFP percentage over 4 passages. BFP percentages were normalized to day 2 (PO) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIG. 21F: Western blots of whole cell extract of NE2 organoids stably overexpressing dTAG-Tip60 and with knockout of endogenous Tip60 using sgTip60_4 (Tip60 dTr4). Actin was used as loading control. FIG. 21G: Western blots of whole cell extract of NE2 Tip60 dTr4 organoids pretreated with dTAGv-l for 4 h and then washed out of dTAGv-l for indicated days. For washout, organoids were extracted from the Matrigel and reseeded in organoid culture conditions without dTAGv-l. FIG.21H: Cell viability of NE2 Tip60 dTr4 organoids after 6 days of treatment with 500 nM dTAGv-l. Data represent mean ± SD from n = 9 biological replicates. FIG. 211: RT-qPCR analysis of Tip60 RNA expression in indicated organoid lines stably expressing dCas9-174932-3629-0421 .1Atty. Dkt. No. 115872-3363KRAB and transduced with control or sgRNA targeting TSS proximal regions of the Tip60 promoter. sgRNA-containing cells were selected by puromycin treatment. Data represent mean ± SD from n = 3 technical replicates. FIG. 21J: Cell competition assays using four independent Tip60 TSS-targeting sgRNAs in indicated organoid lines stably overexpressing dCas9-KRAB, showing relative BFP percentage over 4 passages. BFP percentages were normalized to day 2 (PO) and the NegCtrM at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates.

[0034] FIGs. 22A-22H show that TIP60 acts as a coactivator of MYCL to integrate pro-growth transcriptional outputs of key NEPC TFs and promote lineage survival (Related to FIGs. 17A-17O). FIG. 22A: Top 4 de novo TF motifs enriched at CUT&RUN peaks of indicated TFs in AD2 or NE2 organoids (target coverage > 10%, ranked by p values). FIGs. 22B-22C: UpSet plot showing intersections of significantly downregulated (FDR < 0.05, log2FC < 0) and upregulated (FDR < 0.05, log2FC > 0) genes upon 3-day knockout of indicated factors in NE2 organoids. FIG. 22D: Overrepresentation analyses (FDR < 0.05) of custom prostate epithelial signatures and hallmark gene sets in upregulated genes upon 3- day knockout of indicated genes compared to control sgRNA in NE2 organoids (related to FIG. 17D). FIG. 22E: Cell viability of NE2 Mycl dTr2 organoids (dTAG-Mycl; sgMycl_2) after 6 days of treatment of 500 nM dTAG-13 or dTAGv-l. Data represent mean ± SD from n = 10 biological replicates. FIG. 22F: Average profile showing occupancy (CUT&RUN) of MYCL (via HA tag) and TIP60 at TIP60 peaks in NE2 Mycl dTr2 organoids treated with 500 nM dTAGv-l for 6 h. NT, non-treated. FIG. 22G: Western blots of whole cell extract from NE2 organoids overexpressing indicated Myc, Mycn o Mycl with N-terminal 2xHA- 3xFLAG-dTAG (related to FIG. 17M). Actin was used as loading control. FIG. 22H: Cell viability of NE2 Myc dTrl2 (dTAG-Myc; sgMycl_2) an Mycn dTrl2 (dTAG-Mycn; sgMycl_2) organoids after 6 days of treatment of 500 nM dTAGv-l. Data represent mean ± SD from n = 9 (Myc dTrl2) and n = 7 Mycn dTrl2) biological replicates.

[0035] FIGs. 23A-23K show the acetyltransferase and chromatin reader activities of the TIP60 complex are required for NEPC survival (Related to FIGs. 18A-18R). FIG. 23A: Western blots of whole cell extract from NE2 organoids with Tip60 knockout showing levels of H3, H4 and H2A.Z acetylation. sgRNA-containing cells were sorted out based on BFP positivity 3 days after transduction. Vinculin was used as loading control. FIG. 23B: Western blots of whole cell extract from AD2 organoids with Tip60 knockout. sgRNA- containing cells were sorted out based on BFP positivity 3 days after transduction. In FIGs.184932-3629-0421 .1Atty. Dkt. No. 115872-336323B, 23C, 23D, 23J, and 23K, Actin was used as loading control. FIG. 23C: Western blots of whole cell extract from AD2 organoids with Ep400 knockout. In FIGs. 23B, 23C, 23D, 23J, and 23K, sgRNA-containing cells were selected by puromycin treatment. FIG. 23D: Western blots of whole cell extract from AD2 organoids with Srcap knockout. FIG. 23E: Schematic showing the SRCAP complex. Red outlines indicate subunits with chromatin reader domain(s). Black circles indicate histone modifications reportedly / typically recognized by respective reader subunits. FIG. 23F: Cell competition assays using two independent single or dual sgRNA vectors targeting either or both H2A.Z isoforms in AD2 organoids, showing relative BFP percentage over 4 passages. BFP percentages were normalized to day 2 (PO) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIG. 23G: Cell competition assays using two independent single or dual sgRNA vectors targeting indicated individual TIP60 complex subunits or both paralogs in AD2 organoids, showing relative BFP percentage over 4 passages. Reader domain targeting sgRNAs were utilized for Ing3, Brd8, Yeats4, Mbtdl, Mrgl5 and Mrgx. BFP percentages were normalized to day 2 (PO) and the NegCtrl4 at the respective day (relative %BFP+). Data represent mean ± SD from n = 2 technical replicates. FIG. 23H: Heatmap showing z-score transformed expression (RNA-seq) of subunits of the TIP60 and SRCAP complexes in indicated organoid lines. FIG. 231: Boxplots showing expression (bulk RNA-seq) of indicated subunits of the TIP60 and SRCAP complexes in tumor samples of the SU2C and WCM prostate cancer patient cohorts, p values were calculated using two-tailed Wilcoxon rank sum test. FIGs. 23J and 23K: Western blots of whole cell extract from AD2 organoids with Brd8 (FIG. 23 J) or Yeats4 (FIG. 23K) knockout.DETAILED DESCRIPTION

[0036] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0037] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the disclosure. All the various embodiments of the present disclosure will not be described herein. Many modifications and variations of the disclosure can be made without departing from its spirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatuses within the scope of the disclosure, in194932-3629-0421 .1Atty. Dkt. No. 115872-3363 addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that the present disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0038] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson el al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir ’s Handbook of Experimental Immunology.

[0039] Treatment-related neuroendocrine prostate cancer (NEPC) is an increasingly frequent mechanism of resistance to androgen receptor pathway inhibitor (ARPI) therapy in prostate adenocarcinoma (PRAD). This lineage transition is dependent on upregulation of the NE-specifying transcription factor ASCL1, typically in a genetic background of Rbl and Trp53 loss, which mirrors the high frequency of mutations in these tumor suppressors observed clinically. Here we identify extracellular matrix-integrin-YAPl / TEAD signaling as a critical brake on NEPC lineage transition that prevents ASCL1 upregulation. Deletion of Itgbl, the shared Bl subunit required for collagen and laminin-mediated integrin204932-3629-0421 .1Atty. Dkt. No. 115872-3363 activation, is sufficient to induce ASCL1 and NE lineage gene expression, by activating LATS1 / 2 kinases and subsequent loss inactivation of YAP1 / TEAD signaling. Conversely, restoration of YAP1 / TEAD signaling, by pharmacological LATS1 / 2 inhibition or by expression of constitutively active YAP1 / TAZ mutants, prevents or reverts NEPC lineage transition, in mice and in organoids derived from patients with NEPC. AR cooperates with YAP / TEAD to repress ASCL1, such that their combined inhibition leads to complete reprograming of PRAD into NEPC in vitro, providing a dynamic platform to dissect the molecular events responsible for lineage transition over time. Toward that end, we find that lineage transition is accompanied by a redistribution of the F0XA1 and TEAD cistromes from PRAD to NEPC-specific enhancers that requires the pioneering activity of F0XA1. Collectively, this work shows that extracellular matrix / integrin signaling in the PRAD tumor microenvironment restrains NE lineage plasticity, with clinical implications for perturbation of lineage fate using LATS or TEAD inhibitors. In particular, LATS1 / 2 inhibitors are effective for preventing or reverting NEPC lineage transition, thereby increasing tumor susceptibility to ARSI therapies.

[0040] Further, through characterization of isogenic PRAD and NEPC mouse tumoroid models generated using human-relevant driver mutations (Pten- / -;Rbl- / -;Trp53- / -), we revealed an extensive epigenetic reprogramming in NEPC versus PRAD, particular at enhancers. CRISPR screens focused on TFs and coactivators revealed a NEPC lineage TF survival network comprised of ASCL1, NFIB, SOX1 / 11, FOXA1 / 2 and MYCL with dependency on the TIP60 acetyltransferase complex. During the PRAD-to-NEPC transition, FOXA1, NFIB and TIP60 chromatin binding redistributes to NE specific enhancers, resulting in expression of NE target genes in a coordinate fashion with ASCL1 functioning as a master regulator, culminating in activation of MYCL. MYCL then directly recruits the TIP60 complex to target sites, where it acts as a requisite MYCL coactivator for target gene activation for NEPC growth / survival. The acetyltransferase activity of TIP60 and acetylation of its substrate H2A.Z are both required for NEPC survival, as well as two chromatin reader subunits of the TIP60 complex, BRD8 and YEATS4. Targeted chemical degradation of TIP60 revealed >500-fold dependency in NEPC relative to normal prostate, credentialing the TIP60 complex as a therapeutic target in NEPC, with TIP60 inhibition being effective for preventing or reverting NEPC lineage transition, thereby increasing tumor susceptibility to ARSI therapies.214932-3629-0421 .1Atty. Dkt. No. 115872-3363Definitions

[0041] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and / or separating the subject matter described.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in the present disclosure. Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0043] As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others. “Consisting essentially of’ when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.

[0044] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1.0 or 0.1, as appropriate or alternatively by a variation of + / - 20% or + / - 15%, or alternatively 10% or alternatively 5% or alternatively 2%. As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member.224932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0045] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0046] As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value.

[0047] As used herein, the term “adenocarcinoma” refers to cancer that forms in the glandular tissue, which lines certain internal organs and makes and releases substances in the body, such as mucus, digestive juices, and other fluids. Most cancers of the breast, lung, esophagus, stomach, colon, rectum, pancreas, prostate, and uterus are adenocarcinomas.

[0048] As used herein, the term “administration” of an agent to a subject includes any route of introducing or delivering the agent to a subject to perform its intended function. Administration can be carried out by any suitable route, including, but not limited to, intravenously, intramuscularly, intraperitoneally, subcutaneously, and other suitable routes as described herein. Administration includes self-administration and the administration by another. “Administration” of a cell or vector or other agent and compositions containing same can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated.Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of animals, by the treating veterinarian. In some embodiments, administering or a grammatical variation thereof also refers to more than one doses with certain interval. In some embodiments, the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer. In some embodiments, one dose is repeated for once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. Suitable dosage formulations and methods of234932-3629-0421 .1Atty. Dkt. No. 115872-3363 administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application. In some embodiments, the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer.

[0049] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, / .< ., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In some embodiments, amino acids forming a polypeptide are in the D form. In some embodiments, the amino acids forming a polypeptide are in the L form. In some embodiments, a first plurality of amino acids forming a polypeptide are in the D form, and a second plurality of amino acids are in the L form.

[0050] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.244932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0051] As used herein, the term “analog” refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0052] As used herein, a “cancer” is a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication and in some aspects, the term may be used interchangeably with the term “tumor.” The term “cancer or tumor antigen” refers to an antigen known to be associated and expressed in a cancer cell or tumor cell (such as on the cell surface) or tissue, and the term “cancer or tumor targeting antibody” refers to an antibody that targets such an antigen. In some embodiments, the cancer or tumor antigen is not expressed in a non-cancer cell or tissue. In some embodiments, the cancer or tumor antigen is expressed in a non-cancer cell or tissue at a level significantly lower compared to a cancer cell or tissue. In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, ovarian cancer, melanoma, breast cancer, mesothelioma, renal cancer, or colorectal cancer. In some embodiments, the cancer is a primary cancer or a metastatic cancer. In some embodiments, the cancer is a relapsed cancer. In some embodiments, the cancer reaches a remission, but can relapse. In some embodiments, the cancer is unresectable.

[0053] As used herein, the term “cell population” refers to a group of at least two cells expressing similar or different phenotypes. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells, at least about 10,000 cells, at least about 100,000 cells, at least about 1 x 106cells, at least about 1 x 107cells, at least about 1 x 108cells, at least about 1 x 109cells, at least about 1 x 1010cells, at least about 1 x 1011cells, at least about 1 x 1012cells, or more cells expressing similar or different phenotypes.

[0054] As used herein, “complementary” sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other. In some254932-3629-0421 .1Atty. Dkt. No. 115872-3363 embodiments, the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other. In addition, sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other. For example, a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide. Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch.

[0055] A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a nanoparticle, detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include carriers, such as pharmaceutically acceptable carriers. In some embodiments, the carrier (such as the pharmaceutically acceptable carrier) comprises, or consists essentially of, or yet further consists of a nanoparticle, such as an polymeric nanoparticle carrier or an lipid nanoparticle that can be used alone or in combination with another carrier, such as an adjuvant or solvent. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-264932-3629-0421 .1Atty. Dkt. No. 115872-3363 mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol. A composition as disclosed herein can be a pharmaceutical composition. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0056] As used herein, a “control” is an alternative sample used in an experiment for comparison purpose. A control can be “positive” or “negative.” For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0057] As used herein, the phrase “derived” means isolated, purified, mutated, or engineered, or any combination thereof. For example, an immune cell derived from a donor refers to the immune cell isolated from a biological sample of the donor and optionally engineered.

[0058] As used herein, the term “effective amount” or “therapeutically effective amount” refers to a quantity of an agent sufficient to achieve a beneficial or desired clinical result upon treatment. In the context of therapeutic applications, the amount of a therapeutic agent administered to the subject can depend on the type and severity of the disease or condition and on the characteristics of the individual, such as general health, age, sex, body weight, effective concentration of the engineered immune cells administered, and tolerance to drugs. It can also depend on the degree, severity, and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art.

[0059] As used herein, the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient of a medication, included for the purpose of274932-3629-0421 .1Atty. Dkt. No. 115872-3363 long-term stabilization, bulking up solid formulations, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.

[0060] As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from the same sample following administration of the compositions disclosed herein. The term “expression” also refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription) within a cell; (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation) within a cell; (3) translation of an RNA sequence into a polypeptide or protein within a cell; (4) post-translational modification of a polypeptide or protein within a cell; (5) presentation of a polypeptide or protein on the cell surface; and (6) secretion or presentation or release of a polypeptide or protein from a cell. The level of expression of a polypeptide can be assessed using any method known in art, including, for example, methods of determining the amount of the polypeptide produced from the host cell. Such methods can include, but are not limited to, quantitation of the polypeptide in the cell lysate by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assay and Bradford protein assay.

[0061] As used herein, the term “heterologous nucleic acid molecule or polypeptide” refers to a nucleic acid molecule (e.g., a cDNA, DNA or RNA molecule) or polypeptide that is either not normally expressed or is expressed at an aberrant level in a cell or sample obtained from a cell. This nucleic acid can be from another organism, or it can be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

[0062] As used herein, a "host cell" is a cell that is used to receive, maintain, reproduce and amplify an expression vector. A host cell also can be used to express the polypeptide encoded by the expression vector. The nucleic acid contained in the expression vector is replicated when the host cell divides, thereby amplifying the nucleic acids.284932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0063] As used herein, the term “immune cell” refers to any cell that plays a role in the immune response of a subject. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes. As used herein, the term “engineered immune cell” refers to an immune cell that is genetically modified. As used herein, the term “native immune cell” refers to an immune cell that naturally occurs in the immune system.

[0064] As used herein, the term “increase” or “enhance” means to alter positively by at least about 5%, including, but not limited to, alter positively by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%.

[0065] As used herein, the term “isolated,” “purified,” or “biologically pure” refers to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or polypeptide of the presently disclosed subject matter is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0066] As used herein, the term “isolated cell” refers to a cell that is separated from the molecular and / or cellular components that naturally accompany the cell.

[0067] The term “LATS1 / 2 inhibitor” refers to a compound that is capable of interacting with, and inhibiting the enzymatic activity of LATS1 and LATS2 kinases, which regulate the nuclear localization of YAP and TAZ directly through phosphorylation (pYAPl / pTAZ are cytoplasmic). As used herein, inhibiting LATS1 / 2 enzymatic activity294932-3629-0421 .1Atty. Dkt. No. 115872-3363 means reducing the ability of LATS1 / 2 to phosphorylate a substrate peptide or protein e.g., YAP and TAZ). In some embodiments, the LATS1 / 2 inhibitor reduces LATS1 / 2 enzymatic activity by at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99%. In various embodiments, the concentration of LATS1 / 2 inhibitor required to reduce LATS1 / 2 enzymatic activity is less than about 1 pM, less than about 500 nM, less than about 100 nM, or less than about 50 nM. In some embodiments, the LATS1 / 2 inhibitor is selective, e.g., the LATS1 / 2 inhibitor reduces the ability of LATS1 / 2 to phosphorylate a substrate peptide or protein (e.g., YAP and TAZ) at a concentration that is lower than the concentration of the inhibitor that is required to produce another, unrelated biological effect, e.g., reduction of the enzymatic activity of a different kinase.

[0068] As used herein, the term “ligand” refers to a molecule that binds to a receptor. In particular, the ligand binds a receptor on another cell, allowing for cell-to-cell recognition and / or interaction.

[0069] The term “lymphocyte” refers to all immature, mature, undifferentiated, and differentiated white blood cell populations that are derived from lymphoid progenitors including tissue specific and specialized varieties, and encompasses, by way of non-limiting example, B cells, T cells, NKT cells, and NK cells. In some embodiments, lymphocytes include all B cell lineages including pre-B cells, progenitor B cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, plasma B cells, memory B cells, B-l cells, B-2 cells, and anergic AN1 / T3 cell populations.

[0070] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0071] “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. In some embodiments, a pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a nanoparticle, such as an polymeric nanoparticle carrier or an lipid nanoparticle (LNP). Additionally or alternatively, pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium304932-3629-0421 .1Atty. Dkt. No. 115872-3363 chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They can be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0072] The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0073] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid314932-3629-0421 .1Atty. Dkt. No. 115872-3363 residues are a non-naturally occurring amino acid, e.g., an amino acid analog. The terms encompass amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0074] As used herein, “prevention,” “prevent,” or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0075] As used herein, the term “reduce” means to alter negatively by at least about 5%, including, but not limited to, alter negatively by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%.

[0076] As used herein, a “sample” or “biological sample” refers to a body fluid or a tissue sample isolated from a subject. In some cases, a biological sample may consist of or comprise whole blood, platelets, red blood cells, white blood cells, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and / or chorionic villi, cultured cells, endothelial cells, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid and the like. The term "sample" may also encompass the fluid in spaces between cells, including gingival crevicul ar fluid, bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluids. Samples can be obtained from a subject by any means including, but not limited to, venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art. A blood sample can be whole blood or any fraction thereof, including blood cells (red blood cells, white blood cells or leukocytes, and platelets), serum and plasma.

[0077] As used herein, the term “secreted” in reference to a polypeptide means a polypeptide that is released from a cell via the secretory pathway through the endoplasmic reticulum, Golgi apparatus, and as a vesicle that transiently fuses at the cell plasma membrane, releasing the proteins outside of the cell. Small molecules, such as drugs, can also be secreted by diffusion through the membrane to the outside of cell.324932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0078] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0079] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0080] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0081] As used herein, the term “specifically binds” or “specifically binds to” or “specifically target” refers to a molecule (e.g., a polypeptide or fragment thereof) that recognizes and binds a molecule of interest (e.g., an antigen), but which does not substantially recognize and bind other molecules. The terms “specific binding,” “specifically binds to,” or is “specific for” a particular molecule (e.g., an antigen), as used herein, can be exhibited, for example, by a molecule having a Ka for the molecule to which it binds to of about 10”4M, 10-5M, 1(T6M, 10-7M, 10”8M, 1(T9M, 10l0M, 10 " M, or 10l 2M.

[0082] As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to an individual organism, a vertebrate, or a mammal and may include humans, non-human primates, rodents, and the like (e.g., which is to be the recipient of a particular treatment, or from whom cells are harvested). In certain embodiments, the individual, patient or subject is a human.

[0083] “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0084] As used herein, "synthetic," with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical334932-3629-0421 .1Atty. Dkt. No. 115872-3363 synthesis methods. As used herein, production by recombinant means by using recombinant DNA methods means the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA.

[0085] As used herein, the term “T-cell” includes naive T cells, CD4+T cells, CD8+T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells), activated T cells, anergic T cells, tolerant T cells, chimeric B cells, Regulatory T cells (also known as suppressor T cells), Natural killer T cells, Mucosal associated invariant T cells, and 76 T cells, and antigen-specific T cells.

[0086] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0087] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. Therapeutic effects of treatment include, without limitation, inhibiting recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By “treating a cancer” is meant that the symptoms associated with the cancer are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0088] It is also to be appreciated that the various modes of treatment of diseases as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0089] The compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired344932-3629-0421 .1Atty. Dkt. No. 115872-3363 responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.Lineage Plasticity in Neuroendocrine Prostate Cancer

[0090] The underlying rules and patterns of resistance to targeted therapies are now in transition. After two decades of success in targeting oncogenic drivers (numerous kinases, KRAS, etc.) together with improved next generation inhibitors that eliminate target-based resistance mechanisms (e.g., osimertinib for EGFR-mutant lung cancer), a shift away from “on target” (mutation-based) resistance to “off target” resistance involving changes in cell state - often called lineage plasticity has been observed. Prostate cancer provides a striking example where resistance to ARSIs such as enzalutamide can occur through lineage transition from an epithelial prostate adenocarcinoma (PRAD) to neuroendocrine prostate cancer (NEPC). There is growing evidence of lineage plasticity in other tumors placed under the selective pressure of targeted therapy (lung, pancreas, melanoma, others).

[0091] Lineage plasticity is an epigenetic, potentially reversible process, and occurs only in tumors with a specific genomic mutational context — such as loss of the tumor suppressor genes TP 53 and RBI. Whole exome and RNA sequencing of tumors from a cohort of 500 CRPC patients revealed enrichment for genomic alterations in TP53, PTEN and RBI in tumors with transcriptomic evidence of lineage transition. Through analysis of clinical CRPC at a single cell level, it has been demonstrated that lineage plasticity is even more complex, with up to 10 subtypes of PRAD and 3 subtypes of NEPC revealed by gene regulatory network analysis.

[0092] First, the disease initiates with classic appearing PRAD histology but transcriptional profiling revealed a mixed basal-luminal state with elevated inflammatory pathway signaling. Second, this mixed lineage state subsequently gives rise to four distinct354932-3629-0421 .1Atty. Dkt. No. 115872-3363 tumor-derived lineages defined by master regulator transcription factors (TFs) and their target genes, including NEPC (Ascii), NEPC-like (Pou2f3 gastrointestinal (Gl)-like (Tff3) and mesenchymal (Twist2). Among these, the Ascll+ NEPC subpopulation has the highest proliferation rate and eventually dominates the tumor mass. This transition is accelerated by castration, just as in human NEPC. Two cell-intrinsic kinases (FGFR and JAK) whose activity is upregulated in the mixed basal luminal state and required for subsequent lineage transition to NEPC were identified.LATS1 / 2 Inhibitors

[0093] LATS1 / 2 inhibitors are a class of drugs that block the Large Tumor Suppressor Kinases 1 and 2 (LATS1 / 2), which are core components of the Hippo signaling pathway. Activated LATS1 and LATS2 (LATS1 / 2) then phosphorylate the Yes-associated protein 1 (YAP1) at two key sites, serine 397 and serine 127. When LATS1 / 2 are inactivated, such as under conditions of low cell density, YAP1 is allowed to translocate into the nucleus and associate with the transcriptional enhanced associate domain (TEAD). This triggers a gene expression program responsible for cell growth and proliferation.

[0094] LATS1 / 2 inhibitors can be selective or non-selective, small molecules or biomolecules. Examples of LATS1 / 2 inhibitors include, but are not limited to TDI-011536, VT02956, GA-017, NIBR, 4-(4-(4-(4-Chlorophenyl)piperidin-4-yl)phenyl)-lH- pyrrolo[2,3-b]pyridine, 4-(4-(Piperidin-4-yl)phenyl)-lH-pyrrolo[2,3-b]pyridine, 3-Fluoro-4- (4-(piperidin-4-yl)phenyl)-lH-pyrrolo[2,3-b]pyridine, 4-(4-(3-Fluoro-lH-pyrrolo[2,3- b]pyridin-4-yl)phenyl)piperidin-4-ol, 3-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4- yl)phenyl)piperi din-3 -ol, 3-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)pyrrolidin-3- ol, 3-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)azetidin-3-ol, 4-(4-(3 -Fluoro- 1H- pyrrolo[2,3-b]pyridin-4-yl)phenyl)azepan-4-ol, (lR,5S)-3-(4-(3-Fluoro-lH-pyrrolo[2,3- b]pyridin-4-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol, 6-(4-(3-Fluoro-lH-pyrrolo[2,3- b]pyridin-4-yl)phenyl)-2-azaspiro[3.3]heptan-6-ol, 4-(4-(3-Fluoro-lH-pyrrolo[2,3- b]pyridin-4-yl)phenyl)-3-methylpiperidin-4-ol, (3R,4s,5S)-4-(4-(3-Fluoro-lH-pyrrolo[2,3- b]pyridin-4-yl)phenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)-4-(2-Fluoro-4-(3-fluoro-lH- pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)-4-(2-Chloro-4- (3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)-4- (4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-2-methylphenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-6-methylphenyl)-3,5- dimethylpiperidin-4-ol, (3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-364932-3629-0421 .1Atty. Dkt. No. 115872-33636-methylphenyl)-l,3,5-trimethylpiperidin-4-ol, (3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH- pyrrolo[2,3-b]pyridin-4-yl)-6-methylphenyl)-l-isopropyl-3,5-dimethylpiperidin-4-ol, (3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-6-methylphenyl)-3,5- dimethyl-l-(oxetan-3-yl)piperidin-4-ol, l-((3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH- pyrrolo[2,3-b]pyridin-4-yl)-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-l-yl)ethan- 1-one, 4-((3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-6- methylphenyl)-4-hydroxy-3,5-dimethylpiperidin- 1 -yl)tetrahydro-2H-thiopyran 1 , 1 -dioxide, 6-6 fused Bicyclic Heteroaryl compounds, 2,8-Diazaspiro[4.5]decane compounds, and those disclosed in W02020047037, WO2018198077, WO2022253341, the contents of which are incorporated herein in their entireties. In some embodiments, the LATS kinase inhibitor is selected from among:4932-3629-0421 .1Atty. Dkt. No. 115872-3363TIP60 Inhibitors

[0095] Tip60 is a key member of the MYST family of histone acetyltransferases and involved in a broad spectrum of cellular pathways and disease conditions. It catalyzes the transfer of acetyl groups from acetyl-CoA to specific lysine residues on the TV-terminal tail of nucleosomal core histones. The acetylation of histones results in charge neutralization of lysine residues and decreases the affinity between histones and nucleic acids, and leads to relaxed open chromatin structures.

[0096] TIP60 inhibitors can be selective or non-selective, small molecules or biomolecules. Examples of TIP60 inhibitors include, but are not limited to TH1834, NU9056, Pentamidine, Acetyl CoA, and MG149. In some embodiments, the TIP60 inhibitor is selected from among:Formulations Including TIP60 Inhibitors and / or LATS1 / 2 Inhibitors

[0097] The pharmaceutical compositions of the present technology can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others. Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions.Formulations may optionally contain solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, stabilizers and preservatives, solid binders, lubricants and the like, as384932-3629-0421 .1Atty. Dkt. No. 115872-3363 suited to the particular dosage form desired. In certain embodiments, the compositions disclosed herein are formulated for administration to a mammal, such as a human.

[0098] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, cyclodextrins, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0099] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Compositions formulated for parenteral administration may be injected by bolus injection or by timed push, or may be administered by continuous infusion.

[0100] In order to prolong the effect of a compound of the present disclosure, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and394932-3629-0421 .1Atty. Dkt. No. 115872-3363 crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0101] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents such as phosphates or carbonates.

[0102] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0103] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. In such solid dosage forms the active compound may be404932-3629-0421 .1Atty. Dkt. No. 115872-3363 admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.Methods of Treatment of the Present Technology

[0104] In one aspect, the present disclosure provides a method for treating or preventing prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a LATS kinase inhibitor. In another aspect, the present disclosure provides a method for enhancing responsiveness of a prostate cancer patient or a lung cancer patient to Androgen Receptor Signaling Inhibitor (ARSI) comprising sequentially, simultaneously or separately administering to the patient an effective amount of a LATS kinase inhibitor and an effective amount of an ARSI. In some embodiments, the ARSI is selected from the group consisting of abiraterone acetate, apalutamide, bicalutamide, clascoterone, enzalutamide, flutamide, ARN-509, nilutamide and darolutamide.

[0105] In yet another aspect, the present disclosure provides a method for treating prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a LATS kinase inhibitor and an effective amount of an agent that specifically targets prostate adenocarcinoma (PRAD) or lung adenocarcinoma (LU AD) lineage cells in tumors. In some embodiments, the agent that specifically targets PRAD or LU AD lineage cells binds to STEAP1 or PSMA. Additionally or alternatively, in certain embodiments, the agent that specifically targets PRAD or LU AD lineage cells is an antibody drug conjugate, a T cell engager, a CAR T cell or a radioligand. In some embodiments, the radioligand is lutetium- 177-PSMA-617.

[0106] In any of the preceding embodiments of the methods described herein, the LATS kinase is LATS1 or LATS2. In some embodiments, the LATS kinase inhibitor is selected from among: TDI-011536, VT02956, GA-017, NIBR, 4-(4-(4-(4-Chlorophenyl)piperidin- 4-yl)phenyl)-lH-pyrrolo[2,3-b]pyridine, 4-(4-(Piperidin-4-yl)phenyl)-lH-pyrrolo[2,3- b]pyridine, 3-Fluoro-4-(4-(piperidin-4-yl)phenyl)-lH-pyrrolo[2,3-b]pyridine, 4-(4-(3-414932-3629-0421 .1Atty. Dkt. No. 115872-3363Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)piperidin-4-ol, 3 -(4-(3 -Fluoro- 1H- pyrrolo[2,3-b]pyridin-4-yl)phenyl)piperidin-3-ol, 3-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin- 4-yl)phenyl)pyrrolidin-3-ol, 3-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)azetidin-3-ol, 4-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)azepan-4-ol, (lR,5S)-3-(4-(3- Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol, 6-(4-(3- Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-2-azaspiro[3.3]heptan-6-ol, 4-(4-(3-Fluoro- lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3-methylpiperidin-4-ol, (3R,4s,5S)-4-(4-(3-Fluoro- lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)- 4-(2-Chloro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)-4-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-2-methylphenyl)-3,5- dimethylpiperidin-4-ol, (3S,4s,5R)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)- 6-methylphenyl)-3,5-dimethylpiperidin-4-ol, (3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH- pyrrolo[2,3-b]pyridin-4-yl)-6-methylphenyl)-l,3,5-trimethylpiperidin-4-ol, (3R,4s,5S)-4-(2- Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-6-methylphenyl)-l-isopropyl-3,5- dimethylpiperidin-4-ol, (3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)- 6-methylphenyl)-3,5-dimethyl-l-(oxetan-3-yl)piperidin-4-ol, l-((3R,4s,5S)-4-(2-Fluoro-4- (3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-6-methylphenyl)-4-hydroxy-3,5- dimethylpiperidin-l-yl)ethan-l-one, 4-((3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3- b]pyridin-4-yl)-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-l-yl)tetrahydro-2H- thiopyran 1,1 -di oxide,424932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0107] In one aspect, the present disclosure provides a method for treating or preventing prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a TIP60 inhibitor. In another aspect, the present disclosure provides a method for enhancing responsiveness of a prostate cancer patient or a lung cancer patient to Androgen Receptor Signaling Inhibitor (ARSI) comprising sequentially, simultaneously or separately administering to the patient an effective amount of a TIP60 inhibitor and an effective amount of an ARSI. In some embodiments, the ARSI is selected from the group consisting of abiraterone acetate, apalutamide, bicalutamide, clascoterone, enzalutamide, flutamide, ARN-509, nilutamide and darolutamide.

[0108] In yet another aspect, the present disclosure provides a method for treating prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a TIP60 inhibitor and an effective amount of an agent that specifically targets prostate adenocarcinoma (PRAD) lineage cells or lung adenocarcinoma (LU AD) lineage cells in tumors. In some embodiments, the agent that specifically targets PRAD or LU AD lineage cells binds to STEAP1 or PSMA. Additionally or alternatively, in certain embodiments, the agent that specifically targets PRAD or LU AD lineage cells is an antibody drug conjugate, a T cell engager, a CAR T cell or a radioligand. In some embodiments, the radioligand is lutetium- 177-PSMA-617. In some embodiments, the TIP60 inhibitor is selected from among TH1834, NU9056, Pentamidine, Acetyl CoA, and MG149. In some embodiments, the TIP60 inhibitor is selected from among:434932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0109] Additionally or alternatively, in some embodiments, the ARSI is administered orally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, subcutaneously, intratumorally, topically, by inhalation spray, buccally, or via an implanted reservoir. Examples of ARSIs include, but are not limited to abiraterone acetate, apalutamide, bicalutamide, clascoterone, enzalutamide, flutamide, ARN-509, nilutamide and darolutamide.

[0110] In any and all embodiments of the methods disclosed herein, the prostate cancer or lung cancer harbors a mutation in Rbl and / or Trp53. Additionally or alternatively, in some embodiments, the prostate cancer or lung cancer comprise ASCL1+ cells.Additionally or alternatively, in certain embodiments, the TTP60 inhibitor or the LATS1 / 2 inhibitor is administered orally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, subcutaneously, intratumorally, topically, by inhalation spray, buccally, or via an implanted reservoir.[oni] In any and all embodiments of the methods disclosed herein, the prostate cancer is neuroendocrine prostate cancer (NEPC) or the lung cancer is lung neuroendocrine cancer.

[0112] In any and all embodiments of the methods disclosed herein, the patient is human. Additionally or alternatively, in some embodiments, the patient is non-responsive to at least one prior line of cancer therapy such as chemotherapy.

[0113] Additionally or alternatively, in some embodiments of the methods disclosed herein, the LATS1 / 2 inhibitor or TIP60 inhibitor can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or subsequent to (e.g., 5 minutes, 15444932-3629-0421 .1Atty. Dkt. No. 115872-3363 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the ARSI to the patient.

[0114] In some embodiments, the LATS1 / 2 inhibitor or TIP60 inhibitor and the ARSI are administered to a patient, for example, a mammal, such as a human, in a sequence and within a time interval such that the inhibitor or drug that is administered first acts together with the inhibitor or drug that is administered second to provide greater benefit than if each inhibitor were administered alone.

[0115] For example, the LATS1 / 2 inhibitor or TIP60 inhibitor and the ARSI can be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, the LATS1 / 2 inhibitor or TIP60 inhibitor and the ARSI inhibitor are administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect of the combination of the two inhibitors. In one embodiment, the LATS1 / 2 inhibitor or TIP60 inhibitor and the ARSI inhibitor exert their effects at times which overlap. In some embodiments, the LATS1 / 2 inhibitor or TIP60 inhibitor and the ARSI are each administered as separate dosage forms, in any appropriate form and by any suitable route. In other embodiments, the LATS1 / 2 inhibitor or TIP60 inhibitor and the ARSI are administered simultaneously in a single dosage form.

[0116] It will be appreciated that the frequency with which any of these therapeutic agents can be administered once or more than once over a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 20 days, about 28 days, about a week, about 2 weeks, about 3 weeks, about 4 weeks, about a month, about every 2 months, about every 3 months, about every 4 months, about every 5 months, about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months, about every year, about every 2 years, about every 3 years, about every 4 years, or about every 5 years.

[0117] For example, the LATS1 / 2 inhibitor or TIP60 inhibitor or the ARSI may be administered daily, weekly, biweekly, or monthly for a particular period of time. The LATS1 / 2 inhibitor or TIP60 inhibitor or the ARSI may be dosed daily over a 14 day time period, or twice daily over a seven day time period. The LATS1 / 2 inhibitor or TIP60 inhibitor or ARSI may be administered daily for 7 days.

[0118] Alternatively, a LATS1 / 2 inhibitor or TIP60 inhibitor or ARSI may be administered daily, weekly, biweekly, or monthly for a particular period of time followed454932-3629-0421 .1Atty. Dkt. No. 115872-3363 by a particular period of non-treatment. In some embodiments, the LATS1 / 2 inhibitor or TIP60 inhibitor or ARSI can be administered daily for 14 days followed by seven days of non-treatment, and repeated for two more cycles of daily administration for 14 days followed by seven days of non-treatment. In some embodiments, the LATS1 / 2 inhibitor or TTP60 inhibitor or ARSI can be administered twice daily for seven days followed by 14 days of non-treatment, which may be repeated for one or two more cycles of twice daily administration for seven days followed by 14 days of non-treatment.

[0119] In some embodiments, the LATS1 / 2 inhibitor or TIP60 inhibitor or ARSI is administered daily over a period of 14 days. In another embodiment, the LATS1 / 2 inhibitor or TIP60 inhibitor or ARSI is administered daily over a period of 12 days, or 11 days, or 10 days, or nine days, or eight days. In another embodiment, the LATS1 / 2 inhibitor or TIP60 inhibitor or ARSI is administered daily over a period of seven days. In another embodiment, the LATS1 / 2 inhibitor or TIP60 inhibitor or ARSI is administered daily over a period of six days, or five days, or four days, or three days.

[0120] In some embodiments, individual doses of the LATS1 / 2 inhibitor or TTP60 inhibitor and the ARSI are administered within a time interval such that the two therapeutic agents can work together (e.g., within 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 1 week, or 2 weeks). In some embodiments, the treatment period during which the therapeutic agents are administered is then followed by a non-treatment period of a particular time duration, during which the therapeutic agents are not administered to the patient. This non-treatment period can then be followed by a series of subsequent treatment and non-treatment periods of the same or different frequencies for the same or different lengths of time. In some embodiments, the treatment and non-treatment periods are alternated. It will be understood that the period of treatment in cycling therapy may continue until the patient has achieved a complete response or a partial response, at which point the treatment may be stopped. Alternatively, the period of treatment in cycling therapy may continue until the patient has achieved a complete response or a partial response, at which point the period of treatment may continue for a particular number of cycles. In some embodiments, the length of the period of treatment may be a particular number of cycles, regardless of patient response. In some other embodiments, the length of the period of treatment may continue until the patient relapses.

[0121] In some embodiments, the LATS1 / 2 inhibitor or TIP60 inhibitor and the ARSI are each administered at a dose and schedule typically used for that agent during monotherapy. In other embodiments, when the LATS1 / 2 inhibitor or TIP60 inhibitor and464932-3629-0421 .1Atty. Dkt. No. 115872-3363 the ARSI are administered concomitantly, one or both of the agents can advantageously be administered at a lower dose than typically administered when the agent is used during monotherapy, such that the dose falls below the threshold that an adverse side effect is elicited.

[0122] The therapeutically effective amounts or suitable dosages of the LATS1 / 2 inhibitor or TIP60 inhibitor and the ARSI depend upon a number of factors, including the nature of the severity of the condition to be treated, the particular inhibitor, the route of administration and the age, weight, general health, and response of the individual patient. In certain embodiments, the suitable dose level is one that achieves a therapeutic response as measured by tumor regression or other standard measures of disease progression, progression free survival, or overall survival. In other embodiments, the suitable dose level is one that achieves this therapeutic response and also minimizes any side effects associated with the administration of the therapeutic agent.

[0123] Suitable daily dosages of ARSIs can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of ARSIs are from about 20% to about 100% of the maximum tolerated dose as a single agent. In other embodiments, the suitable dosages of ARSIs are from about 25% to about 90% of the maximum tolerated dose as a single agent. In some embodiments, the suitable dosages of ARSIs are from about 30% to about 80% of the maximum tolerated dose as a single agent. In other embodiments, the suitable dosages of ARSIs are from about 40% to about 75% of the maximum tolerated dose as a single agent. In some embodiments, the suitable dosages of ARSIs are from about 45% to about 60% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of ARSIs are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.

[0124] Suitable daily dosages of LATS1 / 2 inhibitors can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of LATS1 / 2 inhibitors are from about 20% to about 100% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages of LATS1 / 2 inhibitors are from about 25% to about 90% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages of LATS1 / 2 inhibitors are from about 30% to about 80% of the474932-3629-0421 .1Atty. Dkt. No. 115872-3363 maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages of LATS1 / 2 inhibitors are from about 40% to about 75% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages of LATS1 / 2 inhibitors are from about 45% to about 60% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of LATS1 / 2 inhibitors are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.

[0125] Suitable daily dosages of TIP60 inhibitors can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of TIP60 inhibitors are from about 20% to about 100% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages of TIP60 inhibitors are from about 25% to about 90% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages of TIP60 inhibitors are from about 30% to about 80% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages of TIP60 inhibitors are from about 40% to about 75% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages of TIP60 inhibitors are from about 45% to about 60% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of TIP60 inhibitors are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.

[0126] Dosage, toxicity and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.484932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0127] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine useful doses in humans accurately. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0128] Typically, an effective amount of a LATS1 / 2 inhibitor or a TIP60 inhibitor or an ARSI, sufficient for achieving a therapeutic or prophylactic effect, may range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example dosages can be 1 mg / kg body weight or 10 mg / kg body weight every day, every two days or every three days or within the range of 1-10 mg / kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of a LATS1 / 2 inhibitor or a TIP60 inhibitor or an ARSI ranges from 0.001-10,000 micrograms per kg body weight. In one embodiment, LATS1 / 2 inhibitor or TIP60 inhibitor or ARSI concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0129] In some embodiments, a therapeutically effective amount of a LATS1 / 2 inhibitor or a TIP60 inhibitor or an ARSI may be defined as a concentration of a LATS1 / 2 inhibitor or a TIP60 inhibitor or an ARSI at the target tissue of 10'12to 10'6molar, e.g., approximately 10'7molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg / kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single494932-3629-0421 .1Atty. Dkt. No. 115872-3363 daily or weekly administration, but also including continuous administration (e.g., parenteral infusion or transdermal application).

[0130] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.

[0131] The mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some embodiments, the mammal is a human.Kits of the Present Technology

[0132] The present disclosure provides kits comprising one or more TIP60 inhibitors or LATS1 / 2 inhibitors disclosed herein, and instructions for treating or preventing neuroendocrine tumor formation in lung cancer or prostate cancer. When simultaneous administration is contemplated, the kit may comprise a TIP60 inhibitor or LATS1 / 2 inhibitor and an ARSI that has been formulated into a single pharmaceutical composition such as a tablet, or as separate pharmaceutical compositions. When the TIP60 inhibitor or LATS1 / 2 inhibitor and the ARSI are not administered simultaneously, the kit may comprise a TIP60 inhibitor or LATSl / 2 inhibitor and an ARSI that has been formulated as separate pharmaceutical compositions either in a single package, or in separate packages.

[0133] Additionally or alternatively, in some embodiments, the kits further comprise at least one ARSI that are useful for treating or preventing neuroendocrine tumor formation. Examples of ARSIs include, but are not limited to abiraterone acetate, apalutamide, bicalutamide, clascoterone, enzalutamide, flutamide, ARN-509, nilutamide and darolutamide.

[0134] The kits may further comprise pharmaceutically acceptable excipients, diluents, or carriers that are compatible with one or more kit components described herein.Optionally, the above described components of the kits of the present technology are packed in suitable containers and labeled for the treatment or prevention of neuroendocrine tumors. The kits may optionally include instructions customarily included in commercial packages of therapeutic products, that contain information about, for example, the indications, usage,504932-3629-0421 .1Atty. Dkt. No. 115872-3363 dosage, manufacture, administration, contraindications and / or warnings concerning the use of such therapeutic products.EXAMPLES

[0135] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.Example 1: F0XA1 initiates neuroendocrine Unease transition in prostate cancer in response to loss of extracellular matrix-mediated YAP activationMaterials and Methods:

[0136] Organoid culture, analysis and transplantations

[0137] Mouse prostate organoid derivation and culture: Whole mouse prostates, including all lobes, were isolated as previously described. Briefly, dissected prostates were digested with collagenase type II (Gibco) for 2 hours at 37 °C, followed by TrypLE™ Express (Gibco) digestion at 37 °C until a single-cell suspension was obtained. All digestions were supplemented with Y-27632 (10 pM; MedChemExpress, HY-10583) to prevent anoikis, and the resulting cell suspension was filtered through 40-pm strainers to remove debris. Murine prostate organoids were established and maintained under standard culture conditions as previously described. In brief, dissociated epithelial cells were embedded in 20-pL drops of growth factor-reduced Matrigel (Corning, 356231) and overlaid with mouse prostate organoid medium.

[0138] Organoid genetic engineering'. Genetic perturbation in organoids was performed using CRISPR-Cas9 ribonucleoprotein (RNP) complexes as previously described. Briefly, Cas9 protein (IDT) was incubated with synthetic sgRNA (IDT) to assemble the RNP complex prior to nucleofection. A total of 1.2 pM cRNP was used per sgRNA. Dissociated organoid cells (5 x 105- 1 x 106) were resuspended in nucleofection buffer containing RNP complexes and electroporation enhancer (IDT; 1 : 1 molar ratio to RNP) in a final volume of 100 pL. The suspension was transferred to a nucleofection cuvette and electroporated using a Lonza Amaxa Nucleofector II (program T-030). Cells were then centrifuged and seeded into Matrigel for culture. Pten-null organoids were enriched by withdrawal of EGF from the culture medium. For lentiviral transduction, viral titers were determined prior to infection, and a multiplicity of infection (MOI) of 0.2 was used to ensure single-copy integration. A total of 5 x io5cells were used per reaction. cMyc overexpression was achieved by lentiviral transduction of a construct containing cMyc- -IRES- -eGFP GFP-positive cells were514932-3629-0421 .1Atty. Dkt. No. 115872-3363 isolated by fluorescence-activated cell sorting (FACS). For adenoviral transduction, 1 pL of Ad-CMV-Null (Vector Biolabs, 1300) or 1 pL of Ad-CMV-iCre (Vector Biolabs, 1045) was used to infect 5 x io4cells. To enhance infection efficiency, spinoculation was performed at 32 °C and 600 x g for 1 hour.

[0139] RNA isolation, cDNA Synthesis and quantitative PCR'. RNA was isolated from organoids using the RNeasy Plus Mini Kit (Qiagen) according to manufacturer's protocol. RNA concentration was quantified using a NanoDrop (ThermoFisher). Complementary DNA (cDNA) was synthesized using High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific) according to manufacturer’s instructions. Quantitative PCR experiments were conducted on Applied Biosystems QuantStudio 6 Flex Real-Time PCR system. qPCR primers were listed in Supplementary Table.

[0140] Suspension culture'. Organoid cells were dissociated using TrypLE™ Express Enzyme (Gibco) and passed through 40um cell strainers (Fisher Scientific). A total of 1 x 106cells were resuspended in prostate organoid medium and seeded into one well of a Nunclon™ Sphera™ plate (Thermo Scientific, 174932) or an Ultra-low attachment plate (Coming, 3471). Cells were passaged every 3-4 days. For passaging, cells were enzymatically dissociated to single cells using TrypLE™ Express prior to reseeding.

[0141] Protein isolation and western blot analysis'. Organoids were isolated from the Matrigel using cell recovery solution (Coming, 354253). Cells were lysed in RIPA buffer containing protease inhibitors (Calbiochem) and phosphatase inhibitors (Calbiochem) on. Protein concentrations were quantified using a bicinchoninic acid (BCA) assay (Pierce, Thermo Fisher). Lysates were denatured using 4X protein loading dye (SDS 200 nM Tris, 8% SDS, 0.4% bromophenol blue, 40% glycerol, 400 mM 2-mercaptoethanol, pH 6.8). 20- 30 pg of protein was loaded on NuPage 4-12% gradient polyacrylamide gels (Invitrogen). After electrophoresis, protein was transferred to a PVDF membrane and blocked with 5% milk in TBS-T. Primary antibodies were incubated overnight at 4 degrees. Membranes were washed using TBS-T and incubated with secondary antibodies for 1 h at room temperature with shaking. Proteins were visualized using ECL prime (Amersham, GE healthcare) and ImageQuant 800 (Amersham, GE healthcare). Antibodies used in this study are listed in Supplementary Table.

[0142] Orthotopic transplantation'. Organoid cells (2 x 105) were resuspended in 20 pl of a 1 : 1 mixture of growth factor-reduced Matrigel (Corning, 356231) and organoid culture524932-3629-0421 .1Atty. Dkt. No. 115872-3363 medium and injected into the prostate dorsal lobes of immunodeficient NSG mice (J AX 005557) or C57BL / 6J mice (J AX 000664) at 2 months of age.

[0143] Subcutaneous transplantation'. Organoid cells (2 x 105) were resuspended in 100 pl of a 1 : 1 mixture of growth factor-reduced Matrigel (Corning, 356231) and organoid culture medium and injected subcutaneously into the right flank of immunodeficient NSG mice (J AX 005557) or C57BL / 6J mice (JAX 000664) at 2 months of age.

[0144] Generation ofNEPC tumoroids'. Mouse prostate tumors were dissected and finely minced using sterile surgical blades. Tumor fragments were enzymatically dissociated in collagenase type II (Gibco) for 1 hour at 37 °C, followed by further digestion with TrypLE™ Express (Gibco) for 30 minutes at 37 °C. All digestions were supplemented with Y-27632 (10 pM) to prevent anoikis. The resulting cell suspension was passed through 40-pm cell strainers (Fisher Scientific) to obtain a single-cell suspension. Cells were stained on ice for 1 hour with CD51-PE (1 :200; BioLegend, 104106) and EpCAM- Alexa Fluor 647 (1 :200; Abeam, ab237385). CD5 I / EpCAM cells were isolated by fluorescence-activated cell sorting (FACS) and cultured under standard prostate organoid culture conditions.

[0145] Histology and immunostaining

[0146] For GEMM experiments, whole mouse prostates containing all lobes were collected at autopsy. Histological data from all lobes were pooled for quantification. For intraprostatic transplantation and adenoviral infection experiments, injected prostate lobes were collected at autopsy. Prostate tissues were fixed using 4% paraformaldehyde, dehydrated with 70% ethanol, paraffin-embedded and sectioned. H&E staining was performed following standard protocols. Immunohistochemistry and immunofluorescence were performed on a Leica Bond RX automatic Stainer using antibodies listed in Supplementary Table. Formalin-fixed, paraffin-embedded (FFPE) stained tissue sections were scanned using a Pannoramic P250 Flash scanner (3DHISTECH, Hungary) equipped with a 20* / 0.8 NA objective. Whole-slide images were exported as .tif files using SlideViewer software (3DHISTECH, Hungary) for subsequent analysis in Imaged 'FIJI (NIH, USA). For immunohistochemistry (IHC) quantification, color deconvolution was applied to separate hematoxylin and DAB signals. The tissue area and DAB-positive area were determined by thresholding, and the ratio of DAB-positive area to total tissue area was used as a quantitative measure of staining intensity. For immunofluorescence (IF) quantification, multi-channel images were similarly exported and analyzed in ImageJ / FIJI.534932-3629-0421 .1Atty. Dkt. No. 115872-3363Tissue area was defined using the DAPI channel. Nuclei were segmented by generating a DAPI mask followed by watershed separation. Regions of interest (ROIs) approximating individual cells were then used to assess marker positivity, defined by the percent area of signal per marker channel relative to the nuclear ROI.

[0147] Bulk RNA-seq

[0148] RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) from bulk samples and sequenced at the Integrated Genomics Operation Core (MSKCC). cDNA was synthesized from purified RNA using oligo(dT) primers and reverse transcriptase according to standard Illumina protocols. The resulting cDNA was subjected to automated Illumina paired-end library construction. Libraries were sequenced on Illumina HiSeq2000 instruments with paired reads of 100 base pairs (bp) in length per sample, generating approximately 30-40 million reads per sample. Sequence data were processed and analyzed using Partek™ Flow™ software, vl 1.0. Briefly, raw FASTQ files were quality-checked and trimmed to remove low-quality bases and adaptor sequences. Cleaned reads were aligned to the mouse reference genome (mm 10) using the STAR aligner with default parameters. Aligned reads were quantified at the gene level based on Ensembl transcript annotations. Gene-level read counts were normalized using the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) method to account for sequencing depth and transcript length. For visualization, normalized expression values were log2 -transformed. Heatmaps and principal component analysis (PCA) plots were generated using normalized expression values to visualize global transcriptomic differences among samples. Differential gene expression was analyzed using the DESeq2 algorithm (vl.34.0). Genes with an adjusted p < 0.05 and absolute log2 fold change > 2 were considered significantly differentially expressed. Gene set enrichment analysis (GSEA) was performed using the GSEA software (www.gsea-msigdb.org / gsea / index.jsp).

[0149] Bulk RNA-seq analysis on patient cohort

[0150] RNA-seq data from two clinical cohorts were obtained from the cBioPortal for Cancer Genomics (PMID: 23550210). The cohorts included: (1) the SU2C / PCF Dream Team cohort (210 adenocarcinoma and 22 neuroendocrine prostate cancer samples; PMID: 31061129), and (2) the Beltran et al. cohort (Nature Medicine, 2016; 34 adenocarcinoma and 15 NEPC samples; PMID: 26855148).544932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0151] YAP / TAZ target score and Integrin score were calculated using Gene Set Variation Analysis (GSVA) with the single-sample Gene Set Enrichment Analysis (ssGSEA) method (PMID: 19847166), implemented in R (version 4.3.2). The YAP / TAZ target gene set was derived from the art, and the Integrin score was calculated using all annotated integrin genes as the input gene set. Statistical differences in gene set scores and mRNA expression levels of individual genes between adenocarcinoma and NEPC samples were assessed using a two-tailed Wilcoxon rank-sum test.

[0152] Visium spatial transcriptomics

[0153] Data acquisition and pre-processing'. Data were downloaded from GEO accession GSE278936, including spatial alignments, high-resolution histology images, and spot-level gene expression matrices. Each sample was processed independently. After loading the data into Scanpy, low-quality spots (<500 UMIs) and genes expressed in fewer than three spots were filtered using scanpy.pp.filter cells and scanpy.pp.filter genes.

[0154] The expression matrix was then restricted to genes shared between the spatial data and a previously defined single-cell marker gene list. Marker genes were identified from the top 500 differentially expressed genes for each cell state in the single-cell dataset of Zaidi et al. 2024. This list includes canonical markers for neuroendocrine prostate cancer (NEPC), castration-resistant prostate cancer (CRPC), castration-sensitive prostate cancer (CSPC), and stroma, and was used to focus the deconvolution on relevant biological features. Gene counts were deduplicated by summing counts for genes with the same symbol.

[0155] Analysis of Spatial Co-localization'. BayesPrism deconvolution was performed using pybayesprism to estimate the fractional contribution (“theta”) of each cell state to each Visium spot. These theta estimates were used for downstream analyses. A spot was considered NEPC-high if its deconvolved NEPC theta estimate exceeded 0.25. The same threshold was applied to classify spots as CRPC-high, CSPC-high, or stroma-high. We tested a range of thresholds between 0.1 and 0.4, which did not meaningfully affect the results. Spots were classified as mixed if they simultaneously exceeded the 0.25 threshold for both a tumor cell state and stromal content. To assess stromal content in the neighborhood of each spot, we used Squidpy to compute spatial nearest neighbors and calculated the mean stromal theta within a 2-hop radius.

[0156] Single-cell multiome554932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0157] Data pre-processing'. The FASTQ files of the single-cell multiome data were processed by sample with cellranger-arc v. 2.0.2 with alignment against reference genome mmlO. All samples were aggregated using the cellranger-arc aggr function v. 2.0.2. The resulting consensus peak list of genomic regions was annotated with the HOMER1annotatePeaks.pl function version 4.11 with the cellranger-arc refdata-cellranger-arc-mmlO- 2020-A-2.0.0 genes. gtf file.

[0158] Quality control'. Cell filtering criteria were calculated independently for each sample and modality. For single-cell gene expression data, we determined the library size per cell, the number of genes expressed per cell, and the fraction of mitochondrial counts per cell. Specifically, cells expressing fewer than 1,000 or more than 10,000 genes, those with fewer than 1,000 or more than 20,000 total UMI counts, or those exhibiting a mitochondrial read fraction exceeding 20% were excluded. Following this initial cellular filtration, a doublet detection score was computed using Scrublet2v. 0.2.3, employing the parameters min_counts = 2, min_cells = 3, vscore_percentile = 85, n_pc = 50, expected doublet rate = 0.02, sim doublet ratio = 3, and n neighbors = 15. Subsequently, all cells with a doublet score greater than 0.14 were removed. Finally, genes expressed in fewer than 3 cells were discarded. For single-cell chromatin accessibility data, we assessed the minimum library size per cell, the number of peaks detected per cell, the maximum TSS enrichment score, and the nucleosome signal (defined as the ratio of nucleosome-free to mono-nucleosome fragments per cell). Specifically, cells with fewer than 500 or more than 20,000 peaks, or with a total UMI count below 1,000 or above 40,000, were excluded. Additionally, all peaks detected in fewer than 50 cells were removed. Cells were filtered separately for gene expression and chromatin accessibility, with only those cells passing quality control in both modalities being retained. The resulting dataset comprised 23,134 cells, 23,312 genes, and 147,895 peaks.

[0159] Normalization and feature selection'. For single-cell GEX, normalization was performed using a shifted log-transformation of counts, divided by the library size and scaled to 10,000 reads (logCPM+1). For single-cell ATAC normalization, Term Frequency - Inverse Document Frequency (TF-IDF) was computed to weigh the importance of open chromatin regions. Specifically, a shifted log-transformation of the term frequency (TF) was utilized, a method demonstrated to be beneficial in sparse datasets compared to direct TF application, as implemented in the muon3ATAC module. To select highly variable genes, the scanpy4(v. 1.10.3) function pp.compute highly variable was employed with the564932-3629-0421 .1Atty. Dkt. No. 115872-3363‘cellranger’ flavor, resulting in the selection of 3,000 highly variable genes per sample.Highly variable peaks were computed using the scanpy function pp.compute highly variable, with parameters min_mean=0.05 and min_disp=0.5, yielding 16,865 highly variable peaks. Cell cycle genes were scored to determine the cell cycle phase using the scanpy tl.score genes cell cycle function per sample. As a reference, the human cell cycle gene list curated by Tirosh et al.5was converted to align with the mouse gene symbol convention.

[0160] Dimensionality reduction'. For dimensionality reduction, principal component analysis (PCA) was applied to gene expression data, and latent semantic indexing (LSI6) was utilized for chromatin accessibility. A weighted nearest neighbor (WNN) graph, incorporating L2-regularization of distances derived from both modalities on highly variable features, was then computed to generate a joint UMAP.

[0161] Batch correction'. We did not perform any batch correction in the final dataset, because all samples were processed as a single batch.

[0162] Clustering and annotation'. Leiden7clustering, at a resolution of 0.5, resulted in seven distinct clusters. Gene characterization was subsequently performed utilizing a t-test within the scanpy tl.rank genes groups function, employing a one-versus-rest comparison. Differentially accessible peaks were characterized using the Wilcoxon rank-sum test, also within the scanpy tl.rank genes groups function and a cluster-versus-rest setup. The top 20 genes / peaks were then investigated to characterize each cluster. The smallest cluster, comprising 444 cells from various samples, was subsequently excluded. All analyses were performed in Python v. 3.10.15.

[0163] Chromatin profiling and data analysis

[0164] Bulk ATAC-seq Freshly harvested prostate organoid cells were sent for processing. ATAC was performed using 50,000 cells per replicate as previously described (Corces et al. Nature Methods 2017) using the OpenTn5 enzyme (Soroczynski et al, bioRxiv, 2024, PMID: 39026714). The sequencing libraries were purified with SPRIselect magnetic beads (B23318, Beckman Coulter), quantified using a Qubit Flex fluorometer (ThermoFisher Scientific) and profiled using a TapeStation 4200 (Agilent). The libraries were sent for further processing. PicoGreen quantification and quality control by Agilent TapeStation, libraries were pooled and run on a NovaSeq 6000 in a PEI 00 run, using the NovaSeq 6000 S4 Reagent Kit (200 Cycles) (Illumina). The loading concentration was574932-3629-0421 .1Atty. Dkt. No. 115872-33630.5nM and a 1% spike-in of PhiX was added to the run and for quality control purposes. The run yielded on average 50-60M reads per sample.

[0165] CUT&RUN'. Freshly harvested prostate organoid cells were sent for processing. CUT&RUN was performed with IM cells per replicate using the CUTANA™ ChIC / CUT&RUN Kit (Epicypher #14-1048) and the following antibodies: rabbit polyclonal anti-FOXAl (Invitrogen PA5-27157); rabbit monoclonal anti-YAPl (CST, #14074); rabbit monoclonal anti-TEADl (CST, #12292); rabbit anti-H3K4me3 (Epicypher, #13-0041); rabbit anti-mouse IgG (Epicypher, #13-0042). The recovered DNA fragments were quantified and sent to the MSKCC Integrated Genomics Operation core facility for library preparation and sequencing. Immunoprecipitated DNA was quantified by PicoGreen and the size was evaluated by Agilent BioAnalyzer. Illumina sequencing libraries were prepared using the KAPAEvoPrep Kit (Roche 10212250702) according to the manufacturer’s instructions with 0.2-5 ng input DNA and 14 cycles of PCR. Barcoded libraries were run on the NovaSeq 6000 in a PEI 00 run, using the NovaSeq 6000 S4 Reagent Kit (200 Cycles) (Illumina). An average of 5-10M paired reads were generated per sample.

[0166] Sequencing data analysis'. Raw sequencing reads were trimmed and filtered for quality (Q>15) and adapter content using version 0.4.5 of TrimGalore (www.bioinformatics.babraham.ac.uk / projects / trim_galore) and running version 1.15 of cutadapt and version 0.11.5 of FastQC. Version 2.3.4.1 of bowtie2 (bowtiebio, sourceforge.net / bowtie2 / index.shtml) was employed to align reads to mouse assembly mmlO and alignments were deduplicated using MarkDuplicates in Picard Tools v2.16.0. Enriched regions were discovered using MACS2 (github.com / taoliu / MACS) with a p-value setting of 0.001, filtered for blacklisted regions(mitra.stanford.edu / kundaj e / akundaj e / release / blacklists / mm 10- mouse / mmlO.blacklist.bed.gz), and a peak atlas was created using + / - 250 bp around peak summits for ATAC data or using the entire ‘narrowPeak’ region for CUT&RUN data. The BEDTools suite (bedtools. readthedocs. io) was used to create normalized bigwig files. Version 1.6.1 of featureCounts (subread.sourceforge.net) was used to build a raw counts matrix and DESeq2 was employed to calculate differential enrichment for all pairwise contrasts for samples with replicates. For single sample data, MACS2 was run by swapping bams of different conditions to find differential regions. Clusters were discovered by creating a superset of all differential peaks then running k-means clustering from k=2: 10 until cluster redundancy emerged. Peak-gene associations were created by assigning all584932-3629-0421 .1Atty. Dkt. No. 115872-3363 intragenic peaks to that gene, while intergenic peaks were assigned using linear genomic distance to transcription start sites (TSS). Pathway enrichment was calculated by assigning each gene a unique score based on the associated peak with the greatest magnitude change and running GSEA in pre-ranked mode. Motif signatures were obtained using Homer v4.5 (homer.ucsd.edu) on differentially enriched peak regions. Composite and tornado plots were created using deepTools v3.3.0 by running computeMatrix and plotHeatmap on normalized bigwigs with average signal sampled in 25 bp windows and flanking region defined by the surrounding 2 kb. Network analysis was performed using enrichplot::cnetplot in R with default parameters.Supplementary Table - Antibodies594932-3629-0421 .1Atty. Dkt. No. 115872-3363Supplementary Table - sgRNAsSupplementary Table - PCR Primers604932-3629-0421 .1Atty. Dkt. No. 115872-3363614932-3629-0421 .1Atty. Dkt. No. 115872-3363Introduction

[0167] Lineage plasticity - the ability of cancer cells to adopt alternative identities in response to environmental or therapeutic pressures - is increasingly recognized as a major mechanism of resistance to targeted therapies across multiple cancer types, including prostate cancer (7, 2). The mainstay of prostate cancer treatment is androgen deprivation therapy (ADT), to which most patients initially respond. However, resistance inevitably develops, leading to the emergence of castration-resistant prostate cancer (CRPC) (3). To counteract reactivated androgen receptor (AR) signaling in CRPC, next-generation AR pathway inhibitors (ARPIs) such as enzalutamide and abiraterone have become standard of care in CRPC and are now used in earlier treatment settings (4, 5). Despite improved overall survival, an unintended consequence of the selective pressure of ARPI therapy is an increase in treatment-related NEPC, a lineage transition from prostate adenocarcinoma (PRAD) resembling EGFR-mutant lung adenocarcinomas that transition to small cell lung cancer (SCLC) following treatment with EGFR inhibitors (6, 7). Once this lineage switch occurs, NEPC is associated with poor clinical outcomes and is largely unresponsive to existing treatments (S), underscoring the need to understand the molecular events that initiate lineage plasticity, with the goal of preventing or delaying the transition to NEPC.

[0168] Genomic loss of the RBI and TP53 tumor suppressors is highly enriched in human NEPC (and in EGFR-mutant lung cancers that transition to SCLC) and is required for NEPC lineage transition in mouse models (9-12). However, the presence of these mutations alone is not sufficient to initiate lineage programing. For example, RbHTrp53 deficient mouse prostate organoids retain a luminal / basal PRAD lineage when propagated in vitro but undergo lineage transition after in vivo transplantation, suggesting a critical role for the tumor microenvironment (TME) (9, 13). Furthermore, genomically annotated cohorts of CRPC and EGFR-mutant lung cancer reveal that not all adenocarcinomas with RBI and TP53 loss undergo lineage transition in response to therapy (72, 14). The fact that624932-3629-0421 .1Atty. Dkt. No. 115872-3363 treatment-induced lineage transitions can be reversible suggests that epigenetic programs likely play a critical role 15-17). Current evidence supports a model in which Rbl and Trp53 loss establish a chromatin state permissive for lineage switching, but additional tumor cell-extrinsic signals from the TME are needed to initiate and sustain lineage plasticity. Here we explore the nature of these TME cues and how they integrate with tumor-intrinsic programs.Results

[0169] As a first step, we confirmed prior work (9, 13) demonstrating that genetically engineered murine organoids with Rbl and Trp53 loss coupled with cMyc overexpression (RPM) undergo a PRAD to NEPC transition following orthotopic transplantation into mice but fail to do so in organoid culture, as indicated by the absence of staining for the NE lineage marker Ascii (FIGs. 8A-8B). We extended this phenotype to two additional / / i / / 7 / ' 53-deficient genotypes: Rbl4'; Trp534'; Pten4' (TKO) and Rbl4'; Trp534'; Pten4'; cMyc+(TKOM) (FIGs. 8A-8B). These findings document that conventional organoid culture systems fail to fully replicate the in vivo TME necessary for adenocarcinoma-to-NE transition.

[0170] Emergence of NEPC linked to reduced stroma and extracellular matrix: To search for TME factors responsible for this lineage transition, we analyzed histological sections from wild-type normal prostate tissue, as well as PRAD and NEPC regions from the PtRP {Pten4', Rbl4', Trp534', Pb-Cre) genetically engineered mouse model (GEMM). Strikingly, we observed that v / / ?7c / 7 / / / 7-positive fibroblasts, which are sparse in normal prostate tissue and expand in PRAD regions, are relatively depleted in NEPC regions. Similarly, extracellular matrix (ECM) proteins, such as fibronectin and collagen — primarily produced by fibroblasts — are scarce in normal prostate tissue, abundant in PRAD and depleted in NEPC (FIG. 1A). Tumor-associated fibroblasts and collagen were also abundant in the TKO and RPM orthotopic transplantation models at the PRAD stage (2 weeks post-transplantation) but depleted following the transition to NEPC (16 weeks for TKO and 7 weeks for RPM) (FIG. 8C).

[0171] To explore whether a similar depletion of fibroblasts is observed in human NEPC, we analyzed a recently published prostate cancer cohort profiled using Visium spatial transcriptomics {18) for stromal content in regions of PRAD versus NEPC. Using BayesPrism deconvolution to estimate the fractional contribution of PRAD, NEPC and634932-3629-0421 .1Atty. Dkt. No. 115872-3363 stromal cells within each Visium spot (based on theta estimates >0.25, see methods), we identified 5 samples with sufficient intra-specimen PRAD and NEPC content to compare surrounding stromal content. Among PRAD-high spots, 45.2% had high stromal content (35,794 / 79,110) compared to 25.6% of NEPC-high spots (414 / 1618) (FIGs. IB, 1C, and 8D). This pattern of lower stromal content in NEPC-high spots was robust to variations in the theta threshold between 0.1 and 0.4, a result that was independently confirmed through spatial neighborhood analysis. Taken together with the mouse histology, the results suggest that NEPC may preferentially emerge or expand in stroma-poor environments.

[0172] ECM withdrawal initiates PRAD to NEPC lineage transition: Organoids are grown in Matrigel, an ECM preparation containing laminin (60%) and collagen IV (30%), that replicates the stroma-rich TME of PRAD. Because the NEPC transition is associated with depletion of stroma, we considered the possibility that Matrigel prevents the NEPC transition from occurring in vitro. To test this hypothesis, we removed Matrigel by growing TKOM organoids in suspension and monitored Ascii expression as an early marker of NE lineage differentiation. Ascii is not expressed in the luminal adenocarcinoma lineage but is essential for commitment to a neuroendocrine (NE) fate (9, 13). Notably, Ascii protein expression was detected by IHC in a subset of cells within individual organoids after 30 days of suspension culture (FIG. ID). To explore the kinetics of Ascii induction, we performed a time course experiment and documented Ascii mRNA by RT-PCR after 7 days in suspension, with increased levels after 14 days and reaching peak levels at 30 days (FIG. 9A). Of note, Ascii scored as the top upregulated gene in suspension culture by bulk RNA sequencing (RNA-seq) (FIG. IE), among other NEPC-associated TFs, notably Foxa2, Insml, and Sox 2 (FIG. 9B) Furthermore, gene signatures associated with NEPC were enriched in suspension culture while those downregulated in NEPC were depleted (FIG. IF). Thus, removal of Matrigel from TKOM organoids is sufficient to activate a transcriptional program resembling NEPC.

[0173] To explore the dynamics of this phenotypic shift at a single cell level, we performed multi ome (scRNA and scATAC) sequencing of TKOM organoids grown in Matrigel, or in suspension culture for 2 days or 20 days. All cells underwent a substantial transcriptomic shift after 2 days (2 subpopulations) with a further shift into 3 new subpopulations after 20 days (FIG. 1G). Ascii mRNA was detectable after 20 days but not in all cells (FIG. 1H), consistent with the IHC data, and closely matched with changes in chromatin accessibility at the Ascii locus (FIG. II). Foxa2 and Insml were also detectable644932-3629-0421 .1Atty. Dkt. No. 115872-3363 at day 20 in a subfraction of the Ascll+ cells (FIGs. 1J and 9C), suggesting that Ascii expression may precede expression of other NE genes, consistent with its role as a NE lineage master regulator. FOXA1, however, was uniformly expressed at all time points (FIG. 9C)

[0174] ECM-integrin signaling maintains PRAD lineage: While the suspension culture experiments implicate Matrigel (ECM) as a repressor of NE lineage transition in organoid culture, interpretation of these results is confounded by the fact that prolonged growth of epithelial cells in suspension culture can enrich for other phenotypes such as acquisition of stem -like properties (79). The primary constituents of Matrigel, laminin and collagen IV, signal through distinct alpha integrin receptors but share a common beta receptor subunit, integrin beta-1 (ITGB1) (20). To ask if the induction of Ascii seen in suspension culture is a consequence of impaired integrin signaling, we deleted Itgbl in TKOM organoids. Remarkably, Itgbl knockout replicated the mosaic pattern of ASCL1 protein expression observed in suspension culture but now in organoids grown in Matrigel (FIGs. 2A-2B). Itgbl loss also resulted in upregulation of Foxa2 and Insml, as well as the NE markers Chga, Syp and D113 (FIG. 2C). These findings establish that ECM-integrin signaling in prostate organoids is critical to maintain the PRAD lineage state and acts as a brake on the NEPC transition in vitro.

[0175] Having established a critical role of ECM-integrin signaling in PRAD lineage maintenance in vitro, we next considered whether and how this signaling might be disrupted in the in vivo setting. In examining the gene expression changes seen after transfer of TKOM organoids to suspension culture, we noticed that 7 alpha integrins (Itgal,2,3,5,6, 7,v) and 5 beta integrins (Itgbl, 4, 5, 6, 7) were significantly downregulated (FIG. 2D). To determine if this level of downregulation has functional consequences, we replated suspension cells back into Matrigel to reactivate integrin signaling but found that NE marker expression remained stable (FIGs. 2E-2F). Thus, activation of the NE lineage program that follows loss of ECM-integrin signaling cannot be reversed by reengagement with ECM due to sustained downregulation of integrin expression (FIF. 2G). Using an integrin score that quantifies expression of multiple integrin genes, we also observed significantly reduced expression in NEPC versus PRAD tumor cells in PtRP mice (FIG. 2H) and in two human CRPC cohorts with PRAD and NEPC patients (FIG. 21) (72, 27).

[0176] To summarize, ECM-integrin signaling restricts the PRAD to NEPC lineage transition in organoid culture. Once this signal is disrupted, loss of integrin expression by654932-3629-0421 .1Atty. Dkt. No. 115872-3363 tumor cells precludes reversion to a PRAD lineage state, even if abundant ECM is present. These findings support a model where the abundant stromal population associated with the PRAD state in vivo restrains lineage plasticity. Consistent with this model, we find that NEPC emerges in regions with a relative paucity of stromal cells.

[0177] YAP1 / TAZ / TEAD suppresses Ascii induction and NE lineage transition: To elucidate which pathways downstream of integrin engagement suppress the induction of Ascii expression, we first examined canonical kinase signaling events associated with integrin activity (22). pFAK, pSRC and pAKT were attenuated when cells were cultured in suspension or following Itgbl knockout, confirming that the circuitry of integrin signaling that has been well defined in other cell types is conserved in prostate organoid culture (FIGs. 3A-3B). We also examined the status of the YAP1 / TAZ / TEAD (Hippo) pathway (hereafter called YAP / TEAD), a target of integrin signaling linked to mechanical sensing (ECM stiffness). Loss of integrin engagement resulted in reduced YAP / TEAD signaling, as measured by increased pYAPl (a substrate for LATS1 / 2 kinase) and reduced expression of the YAP / TEAD target gene CYR61. To determine which, if any of these signaling events are involved in Ascii upregulation, we used a panel of small-molecule inhibitors targeting FAK, SRC, PI3K, MEK or the YAP / TEAD complex to test if any might reproduce the Ascii induction phenotype seen with Itgbl KO. Of the kinase inhibitors, only the Src family inhibitor dasatinib gave a reproducible (~30-fold) increase in Ascii expression. More striking, however, was a ~300-fold increase in Ascii seen with IAG933, which selectively disrupts the YAP1 / TEAD protein-protein interaction (FIG. 3C) (23). IAG933 (hereafter called TEADi) also blocked expression of the YAP / TEAD target gene Ctgf and Cry61 (-90% decrease), as did dasatinib (-50% decrease), consistent with the known role of SRC kinases in activating YAP / TEAD through LATS1 / 2 inhibition (FIG. 3D) (24).

[0178] To build on the pharmacologic evidence implicating YAP / TEAD as a critical regulator of Ascii induction, we turned to genetic experiments using gain and loss of function approaches.

[0179] First, we generated RPM and TKOM mouse prostate organoids in a Yap l^A / Wwtr l^A background (the Wwtrl gene encodes TAZ) to ask whether deletion of these TEAD co-activators would activate NE gene expression. We observed >100-fold induction oh Ascii and upregulation of other NE genes within 6-10 days of YapHWwtrl codeletion (by adenoviral Cre infection) (FIG. 3E), as well as the expected loss of YAP / TEAD target gene expression (Cyr61, Ctgf, Axl, Ptpnl4) (FIG. 10A). YapHWwtrl co-664932-3629-0421 .1Atty. Dkt. No. 115872-3363 deletion also resulted in downregulation of several alpha (Itga2, Itga5, Itgav) and beta integrin genes (Itgb4, Itgb5, Itgb6) (FIG. 10B), mirroring the loss of integrin expression seen earlier when YapHWwtrl intact organoids were placed in suspension culture (FIG. 2D) Thus, YAP / TEAD is a key regulator of integrin expression in this context. Conversely, expression of a constitutively active mutant of either YAP (YAP5SA) or TAZ (TAZ4SA), both of which are resistant to inactivation by LATS1 / 2 phosphorylation, blocked the induction of Ascii mRNA and other NE markers (17oxa2, Insml, Chgd) when RPM and TKOM organoids were placed in suspension culture, while maintaining YAP / TAZ target gene expression (Cyr61, Ctgf Axl, Ptpnl4) (FIGs. 3F and IOC).

[0180] Collectively, the organoid experiments provide evidence that ECM-integrin engagement reinforces PRAD lineage identity through YAP / TEAD activation. As discussed earlier, PRAD to NEPC lineage plasticity in vivo is associated with a transition from a stroma rich (high ECM) to a stroma poor (low ECM) TME (FIGs. 1A-1B). To determine whether these in vivo TME changes are also linked to YAP / TEAD activity, we performed multiplex immunofluorescence (IF) on prostate tissue sections from 9-week-old PtRP mice, when NEPC first starts to emerge from PRAD. Using phosphoFAK (pFAK) as a readout for ECM-integrin engagement, we found that loss of pFAK in tumor cells was highly correlated with reduced nuclear YAP1 staining and gain of ASCL expression as measured by multiplex immunofluorescence (p<0.005) (FIGs. 3G-3H). Furthermore, YAP1 / TEAD activity in human CRPC cohorts (via RNA signatures) was significantly reduced in NEPC compared to PRAD (FIGs. 3I-3K), consistent with scRNA-seq data from a smaller CRPC cohort (25). In sum, evidence from organoids, GEMMs and human CRPC supports a model whereby ECM-integrin engagement plays a role in maintenance of the PRAD lineage through sustained YAP / TAZ / TEAD signaling.

[0181] LATS inhibition impairs acquisition and maintenance of neuroendocrine state: Earlier we showed that the PRAD to NEPC lineage transition following loss of ECM- integrin engagement cannot be reversed by re-exposure to ECM - likely due to loss of integrin expression (FIGs. 2E-2G). However, the fact that YAP / TEAD activity is regulated by LATS 1 / 2 kinase activity (YAP1 and TAZ phosphorylation by LATS 1 / 2 results in cytoplasmic retention and inability to bind TEAD (26)) (FIG. 4A) led us to ask whether reduced YAP / TEAD activity caused by loss of ECM-integrin engagement could be restored through LATS kinase inhibition. Indeed, treatment of RPM and TKOM organoids with the LATS 1 / 2 kinase inhibitor TRULI (27) blocked YAP1 phosphorylation induced by674932-3629-0421 .1Atty. Dkt. No. 115872-3363 suspension culture (within 4 hours) and restored expression of the canonical YAP1 / TEAD target genes such as CRY61 (FIG. 4C). More importantly, restoration of YAP / TEAD activity prevented the induction of Ascii and other NE markers (Foxa2, Insml. Chgd) following 14 days of suspension culture, providing proof of concept toward a potential pharmacological approach to prevent the PRAD to NEPC transition (FIGs. 4D and 11 A).

[0182] We next asked whether LATSi can reverse an already established NEPC phenotype by first establishing ASCL1+ tumoroids from NEPC tumors that developed in vivo following orthotopic transplantation of RPM and TKO organoids (see methods for details) (FIGs. 11B-11E). LATSi blocked YAP1 phosphorylation and restored YAP / TEAD activation, as shown by induction of target genes such as CYR61 and PTPN14 (FIG. 4E). Remarkably, after 4 days of treatment, these NEPC tumoroids had near complete loss of ASCL1, INSMI and CHGA expression and gained expression of PRAD lineage markers such as CK8, TROP2 and the API complex proteins FOSL2 and JUNB (FIG. 4E).

[0183] Having shown that the PRAD to NEPC lineage transition can be blocked or reversed by LATSi in mouse tumoroid models, we asked whether this reversion can extend to human NEPC. Using patient-derived organoids (PDOs) established from CRPC patients with NEPC (2S, 29), we found that LATSi activated YAP / TEAD target gene expression (CRY61, CTGF) in a concentration-dependent manner, confirming the same pathway is intact in human CRPC models (FIG. 4F). 10 days of LATSi treatment robustly restored YAP1 nuclear protein expression, coupled with a reduction in the percentage of ASCL1 expressing cells from >60% to <5% (FIGs. 4G-4H), further credentialling LATS1 / 2 kinase as a therapeutic target to prevent or reverse PRAD to NEPC lineage plasticity.

[0184] Unfortunately, we were unable perform equivalent experiments in mice using TRULI or the chemically related LATSi TDI-011536 (30) due to rapid in vivo clearance with full recovery of pYAP in prostate tissue 4 hours after drug administration (FIG. HF). We therefore turned to a genetic approach, using the previously described constitutively active YAP15SAallele (FIG. 3F) but now in a doxycycline (Dox) inducible format such that we could maintain YAP activation in RPM organoids during the time interval when the PRAD to NEPC transition occurs. Remarkably, sustained induction of YAP15SA(or wildtype YAP1) starting 2 weeks after transplantation prevented the appearance of any detectable ASCL1+ cells in PRAD tumors that emerged 21 days later, whereas tumors that developed from RPM organoids transduced with the Dox empty vector control (EV) had abundant regions of ASCL1+ NEPC (FIGs 4I-4J).684932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0185] PRAD to NEPC lineage transition in vitro-. The above data provide clear evidence that disruption of YAP / TEAD activity (through suspension culture, Itgbl deletion or pharmacologic blockade of YAP / TEAD binding) promotes NE lineage transition. However, in situ analysis of ASCL1 protein expression reveals a mosaic pattern with all three methods of YAP / TEAD perturbation (FIGs. ID, 2A, and 5A) suggesting that other regulatory pathways may play a role. In addition, the mRNA levels of Ascii and other NE markers (Foxa2, Insml. Ncam) achieved in organoids following YAP / TEAD perturbation do not reach the level seen in NEPC tumors that develop in organoids transplanted in vivo (FIG. 12)

[0186] PRAD organoids may undergo progressive reprograming to a complete NEPC lineage state. To address this possibility, we compared the transcriptomes of PRAD organoids treated with vehicle control (DMSO), single inhibitors (TEADi or ARi) and doublet combination (TEADi / ARi) in a time course experiment. We harvested organoids at 8 days to capture an intermediate state of lineage reprogramming and at 42 days to represent the final NE lineage state achieved in vitro. As an additional control, we included three independently derived NEPC tumoroids (NE1-3) to compare the NEPC state generated by in vitro reprogramming with the fully reprogramed NEPC state that develops in vivo.

[0187] Using principal component analysis (PCA) to display the entirety of the transcriptomic data, we found that each single treatment, and the doublet clustered relatively closely with the DMSO control, indicative of modest cell state changes when assessed at the level of the whole transcriptome (FIG. 5B). In stark contrast, all three NE tumoroids clustered at the other extreme of the PCA plot showing the full NEPC transition seen in vivo. To verify that the extremes of the PCA plot do indeed reflect the PRAD and NEPC states respectively western blotting was performed with PRAD and NEPC samples for expression of NE markers at the protein level (ASCL1, FOXA2, INSMI), with the results confirming the PCA plot (FIG. 5C). In sum, these data establish an organoid platform for complete reprogramming from PRAD to NEPC through combined and sustained inhibition of TEAD and AR.

[0188] FOXA1 is implicated in the PRAD to NEPC lineage transition: To explore the specific molecular events responsible for the PRAD to NEPC lineage transition, we initially focused on the YAP1 / TEAD axis, using CUT&RUN to map the chromatin binding sites of YAP1 and TEADI across the PRAD and NEPC lineages. Focusing initially on TEADI peaks, we observed a remarkable relocalization of binding sites when comparing the PRAD694932-3629-0421 .1Atty. Dkt. No. 115872-3363 state to the NEPC state (FIG. 6A). In PRAD, genes and pathways enriched at the sites of TEAD peaks include actin filament organization, epithelial morphogenesis, and cell substrate adhesion, all consistent with an adenocarcinoma lineage (exemplified by Tacstd2). Conversely, genes and pathways enriched at the sites of the redistributed TEAD peaks in the NEPC state include regulation of neurogenesis, forebrain development and synapse pathways (exemplified by Ascii) (FIGs. 6B-6D).

[0189] YAP1 peaks in the PRAD state overlapped precisely with TEAD1 peaks, as expected, since these two proteins form a complex, but were absent in the NEPC state because LATS kinase activation disrupts the YAP / TEAD complex (FIG. 6A). The fact that robust TEAD peaks are now detected at the enhancers of NEPC-specific genes in the absence of YAP raises the question whether TEAD binding plays a role in their expression. To address this, we performed CRISPR experiments targeting TEAD1, the most abundantly expressed of the four TEAD family members in the NEPC state, and achieved robust knockdown of TEAD 1 expression, albeit with a low level of residual TEAD expression detected using a pan TEAD antibody presumably from other TEAD family members (FIG. 13A). Despite this caveat, Ascii, Chga and Ncaml mRNA and protein were reduced by -50% following TEAD1 knockdown (FIG. 13B), evidence that the relocalized TEAD peaks seen with the NEPC transition play a role in maintenance of NE-specific gene expression. TEAD knockdown lowered the magnitude of Ascii induction by -50% (FIG. 13C), similar to the effect in established NEPC organoids (FIG. 13B). Importantly, this TEAD-dependent gene regulation occurs in the absence of coactivation by YAP or TAZ. Because TEADs are DNA binding proteins without intrinsic coactivation function, it will be of interest to determine whether another (perhaps NE-specific) coactivator is recruited to perform this function.

[0190] To further investigate the biology underlying the NEPC-specific TEAD1 peaks, we performed motif analysis. TEAD binding motifs were enriched in both PRAD and NEPC lineages, but there were clear lineage-specific differences in co-enriched motifs, specifically AP-1 sites in PRAD and FOXA and ASCL1 sites in NEPC (FIG. 6E). The fact that FOXA motifs are co-enriched at the NEPC-specific TEAD binding sites raises the possibility that FOXA1, which is abundantly expressed in both PRAD and NEPC states, plays a role in the lineage transition. To explore this possibility, we first performed CUT&RUN and confirmed that FOXA1 peaks are indeed present at the NEPC-specific TEAD binding sites (FIG. 6A).704932-3629-0421 .1Atty. Dkt. No. 115872-3363Remarkably, F0XA1 peaks re-localize from PRAD to NEPC specific enhancers, similar to the pattern seen with TEAD.Discussion

[0191] Next generation ARPIs extend prostate cancer survival through increased selective pressure on AR. Despite this clinical success, tumor cells can escape AR dependence by transitioning from AR-dependent luminal cells to AR-independent NEPC cells (lineage plasticity). Here we show that the YAP / TEAD pathway plays a key role in maintenance of the PRAD lineage state, precluding tumor cells from undergoing the PRAD to NEPC lineage switch. Prior work on Hippo pathway signaling, initially in flies and subsequently in mammalian cells, has established a critical role of YAP / TEAD in growth regulation (26, 35-37). In the context of tumor cells, the role of YAP / TEAD in growth regulation is more nuanced. Studies with pharmacologic inhibitors reveal YAP / TEAD dependency in cancers with Hippo pathway mutations (such as mesotheliomas with NF2 loss) but not more broadly, even in tumors with robust YAP / TEAD activation (23, 38). Rather, we provide evidence here that YAP / TEAD functions as a gatekeeper, preventing epithelial tumor cells at risk for lineage plasticity from undergoing a lineage transition. Using PRAD as a model, we find ECM engagement is the primary mechanism of YAP / TEAD activation, consistent with prior evidence linking YAP / TEAD to integrins and mechanical stress (39). Once PRAD tumor cells lose contact with ECM (by experimental withdrawal in organoid culture or stochastically during in vivo tumor expansion), YAP / TEAD activity is acutely switched off, followed by induction of ASCL1 and an eventual transition to NEPC. Importantly, this switch cannot be easily reversed by reengagement with ECM because integrin expression (which itself is YAP / TEAD dependent) is lost. Thus, tumor cells poised for lineage transition (i.e., those with RB loss) enter a point of no return once the YAP / TEAD signal is switched off.

[0192] We also find that ARPI therapy, the therapeutic intervention that initiates the NEPC transition in CRPC patients, contributes to the PRAD to NEPC transition in our organoid model, particularly when YAP / TEAD has already been impaired. The mechanism is cell autonomous because the NE lineage transition can be fully elicited in cultures that only contain tumor cells. A plausible explanation is that AR plays a role in maintenance of luminal lineage identity, even in CRPC. That said, non-cell autonomous mechanisms may also be at play. Intriguingly, ECM production (e.g., collagen) by stromal cells in the normal mouse prostate is AR-regulated (40). One consequence of systemic ARPI therapy could be714932-3629-0421 .1Atty. Dkt. No. 115872-3363 reduced YAP / TEAD activation in tumor cells due to reduced ECM production by AR- positive stromal cells, thereby lowering the threshold for NEPC transition to occur.

[0193] Our ability to fully recapitulate the PRAD to NEPC transition in vitro provided an opportunity to investigate the molecular details of this dynamic process at a level of resolution not easily achievable in vivo. Toward that end, we first examined the TEAD chromatin binding of across the genome and uncovered a striking redistribution of TEAD peaks from PRAD-specific to NEPC-specific enhancers. Equally striking was the parallel redistribution of FOXA1 peaks, raising the intriguing possibility that FOXA1, a pioneer factor expressed in both PRAD and NEPC, plays a pivotal role in initiating the lineage transition (FIG. 7).

[0194] The fact that lineage plasticity in our models can be reversed by restoring YAP / TEAD activity through LATS kinase inhibition (or genetically through constitutive YAP activation) demonstrates that CRPC patients at risk for plasticity could benefit from treatment with a clinical grade LATS inhibitor. Such an intervention would not only reinforce PRAD lineage maintenance but also reprogram tumors with mixed lineage phenotypes (an increasingly common clinical phenotype) to a homogeneous PRAD state. Treatment options could start with transient LATS inhibition followed by treatment with definitive PRAD therapy. Novel ARPIs are one option, and emerging treatments using radioligands, antibody drug conjugates or T cell engagers directed at PRAD-specific cell surface antigens (such as PSMA or KLK2) are another possible route.

[0195] The data shown herein demonstrates that LATS kinase inhibition reprograms tumors to PRAD state or prevents the conversion from PRAD to NEPC state. Accordingly, the methods of the present disclosure are effective for treating or preventing prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a LATS kinase inhibitor and for enhancing responsiveness of a prostate cancer patient or a lung cancer patient to Androgen Receptor Signaling Inhibitor (ARSI) comprising sequentially, simultaneously or separately administering to the patient an effective amount of a LATS kinase inhibitor and an effective amount of an ARSI.Example 2: The TIP60 acetyltransferase complex is a critical dependency in neuroendocrine prostate cancer through its role as a transcriptional coactivator of MYC family oncogenesMaterials and Methods724932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0196] All commercially available products, kits, and instruments were used per the manufacturer’s protocol or a standard protocol except where stated otherwise.

[0197] Organoid culture. Mouse normal prostate and tumor organoids and human PDOs were cultured under standard conditions as previously described herein.

[0198] Subcutaneous and orthotopic transplantation. Subcutaneous and orthotopic transplantation were performed using dissociated organoids following standard protocols.

[0199] Establishment and culture of isogenic tumor organoid series. Pten, Rbl, Trp53 TKO NEPC and PRAD tumors were derived using an organoid transplantation based tumor formation and lineage transformation system, with luminal 2 prostate epithelial cells which we observed to have highest efficiency for NEPC transition compared to luminal 1 or basal cells. Ptenfl / fl; Rblfl / fl; Trp53fl / fl; RosamT / mGluminal 2 prostate epithelial cells were isolated via FACS and utilized to establish normal prostate organoid culture (WT). Normal prostate organoids were transduced with Adeno-Cre to induce recombination and deletion of the floxed tumor suppressors. TKO cells (GFP+) were purified via FACS to establish organoids (TKO-AD1). TKO organoids were further transplanted subcutaneously into mice to allow tumor development. Tumors were harvested after 3-4 months, which showed mixed histology including NEPC and PRAD. Tumor organoids were derived from dissociated TKO tumors and then histological phenotypes and marker expression were determined. T61 (PRAD), T70A (NEPC) and T101A (NEPC) were established as stable, pure PRAD or NEPC organoid lines through subcloning and serial passage.

[0200] Organoid engineering. To establish organoid lines with stable transgenic expression, organoids were infected with lentivirus encoding gene of interest. Genetic knockout was performed using CRISPR-Cas9 technology. Organoids stably expressing lenti-Cas9-blast was infected with lentiviral sgRNA.

[0201] Histology and immunostaining. H&E staining was performed using standard protocols. Immunofluorescence and immunohistochemistry were performed on a Leica Bond RX automatic Stainer. All formalin-fixed paraffin-embedded stained tissue was scanned using a MIRAX scanner.

[0202] Flow cytometry. Intracellular staining of DLL3 was performed using BDCytofix / Cytoperm Fixation / Permeabilization Kit (BD Biosciences). Flow cytometric analyses for cell competition assay were performed using a BD Fortessa instrument. Cell sorting was performed using a SONY MA900 instrument with lOOum sorting chips.734932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0203] Whole cell extract preparation and western blotting. Whole cell extract was prepared using TOPEX buffer as previously described. Organoid were lysed at room temperature for 10 min and protein concentration was determined by BCA assay (Thermo Fisher). Samples were boiled in Laemmli buffer (Bio-rad) at 70 C for 10 min for immunoblotting following standard protocols.

[0204] Growth assay. Growth assays were performed using CellTiter Gio (Promega) following manufacture’s protocol. Organoids were seeded in round bottom 96-well dishes and cultured for 6 days before viability assay.

[0205] Immunoprecipitation. Nuclear extracts from organoids were prepared using the Dignum protocol and incubated with FLAG M2 beads overnight. Beads were washed and proteins were analyzed using western blotting.

[0206] ATAC-seq. ATAC-seq was performed as previously described with Tn5 transposase (Illumina). Transposed genomic DNA was purified using a Zymo DNA Clean and Concentrator- 5 Kit and subjected to PCR amplification using NEBNext High-Fidelity 2x PCR Master Mix with custom Nextera PCR primers ordered from IDT. Libraries were sequenced with an Illumina NextSeq 550 instrument.

[0207] CUT&RUN. CUT&RUN was performed as previously described using in-house purified pAG-MNase and indicated antibodies. CUT&RUN library was prepared using NEBNext Ultra II DNA Library Prep Kit (NEB) according to manufacturer’s protocol and sequenced with an Illumina NextSeq 550 instrument.

[0208] RNA-seq. Total RNA was extracted using RNeasy Plus Mini Kit (Qiagen) according to manufacturer’s protocol. Library was prepared using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) according to manufacturer’s protocol. Libraries were sequenced with an Illumina NextSeq 550 instrument.Introduction

[0209] Lineage plasticity, a process by which tumor cells undergo cell state changes in response to various stresses during cancer progression, is an emerging hallmark of cancer. Lineage plasticity contributes to cancer progression and therapy resistance, exemplified by a well-documented, clinically important and molecularly converged transition from adenocarcinoma to neuroendocrine histology in both prostate and lung cancers. It serves as an emerging, aggressive mechanism to evade increasingly potent targeted therapies by744932-3629-0421 .1Atty. Dkt. No. 115872-3363 assuming an alternative lineage fate that is completely independent of the original oncogenic drivers such as AR and EGFR. Neuroendocrine carcinomas can also emerge de novo across multiple epithelial tissues and display conserved molecular signatures and subtypes. Notably, neuroendocrine malignancies are highly aggressive with no effective treatment options, hence representing a major clinical challenge. Here, we use NEPC as a model to investigate cancer lineage plasticity and reveal novel lineage biased vulnerabilities associated with the neuroendocrine phenotype.

[0210] Genomic and transcriptomic analyses have shown that except Rb loss there is an overall lack of NEPC-specific genetic alternations compared to castration resistant PRAD. Nevertheless, NEPC and its molecular subtypes can be clearly defined by expression of key transcription factors. Previous candidate-based studies have functionally implicated several TFs (ASCL1, FOXA1 / 2, SOX2, MYCN, BRN2, ONECUT2, PROXI, NKX2-1 etc.) in prostate cancer neuroendocrine transformation and maintenance of the neuroendocrine phenotype and / or NEPC survival, which revealed remarkable similarity in TF dependencies between NEPC and SCLC. However, the full repertoire of NEPC dependency TFs and their regulatory mechanisms / hierarchy remains to be further elucidated. Furthermore, epigenomic profiling in patient derived systems has revealed distinct chromatin landscape in NEPC vs. PRAD, e.g. chromatin accessibility, H3K27ac modification, and 3D architecture, suggesting epigenetic mechanisms are associated with NEPC transformation. Nevertheless, the chromatin enzymes and machineries underlying such ‘epigenetic mechanisms’ remain poorly defined. Chromatin regulators under intensive clinical investigations such as epigenetic repressors EZH2, DNMTs, HDACs and LSD1 and activators BRD4 and NSD2 have been implicated as potential targets for NEPC. However, most of them also have reported functions in PRAD and thus their lineage specificity remains incompletely defined. Moreover, more unbiased functional studies on lineage specific requirements of epigenetic modifiers are currently lacking and therefore the roles of the majority of chromatin regulators in NEPC tumorigenesis remained unknown. Therefore, there is an unmet need to identify chromatin modifier vulnerabilities and their mechanistic basis in NEPC.

[0211] Transcriptional coactivators are usually large multi-subunit complexes or multidomain proteins that contain enzymatic (ATP-dependent chromatin remodeling / post- translational modification) and chromatin binding (reader) modules, making them appealing candidates for pharmacologic perturbation. Furthermore, recent studies showed that different TFs recruit distinct sets of coactivators in regulating the multi-step transcriptional754932-3629-0421 .1Atty. Dkt. No. 115872-3363 process and that different types of enhancers and promoters also display distinct coactivator requirements, highlighting that coactivator functions are genomic-context dependent. These observations also led to an intriguing question whether differential TF expression and epigenomic landscape, e.g. accessibility and covalent modifications, upon NEPC transformation confer new, NEPC lineage-biased coactivator dependencies, which could be therapeutically exploited to treat lethal NEPC tumors.

[0212] However, addressing these questions in the context of lineage plasticity requires defined isogenic models of each lineage state for perturbation and stringent comparison. GEMMs are intrinsically low-throughput and patient derived systems are genetically heterogenous. The lack of scalable, in vitro systems that closely mimic in vivo lineage states has been a barrier for the detailed biochemical analyses required for a more precise molecular understanding. To this end, we developed isogenic tumor-derived organoid models capturing PRAD and NEPC stages of prostate cancer lineage transition. Through comprehensive epigenome profiling, we demonstrated dramatic enhancer reprogramming among functional chromatin compartments. Using TF and coactivator-focused CRISPR screening and chemical genetic targeted protein degradation, we further identified five key families of NEPC lineage survival TFs and the TIP60 acetyltransferase complex as a critical NEPC-biased coactivator dependency. The elevated TIP60 dependency emerges in NEPC with the acquired addiction to activities of MYC family oncogenes, which requires its coactivator function, and reprogrammed chromatin landscape, which collectively drives a profound TIP60 genomic redistribution. Importantly, the TIP60-MYCL complex integrates pro-growth oncogenic transcriptional output from multiple essential NEPC TFs. Moreover, we revealed that TIP60 acetyltransferase activity is essential for NEPC, and identified reader subunits required for its genomic occupancy, biochemical function, and ability to sustain NEPC survival. Therefore, our study credentials the TIP60 complex as a therapeutic target in NEPC.Results

[0213] The following materials discuss the data shown in FIGs. 14A-23K.

[0214] Isogenic organoid models reveal epigenetic reprogramming during prostate cancer neuroendocrine transformation. To identify dependencies specifically acquired after PRAD transitions to NEPC, we leveraged our earlier work using genetically modified primary prostate organoids to model this transition in vivo (Romero et al 2024).764932-3629-0421 .1Atty. Dkt. No. 115872-3363Specifically, using tumor explants from organoid-transplanted mice, we derived several isogenic PRAD and NEPC tumoroid pairs, then expanded these tumoroids to a scale amenable for extensive biochemical characterization and CRISPR screening (see methods) (FIG. 14A). Histologic and immunohistochemical (IHC) characterization of these isogenic pairs confirmed retention of the morphological features and lineage marker expression seen in PRAD (ECAD, AR) and NEPC (ASCL1, SYP and DLL3) respectively.

[0215] Transcriptomic profiling showed dramatic gene expression changes in NEPC vs. PRAD organoids highly reminiscent of tumors of the TKO GEMM upon NEPC transition, e.g. downregulation of luminal / AR pathway genes and inflammatory JAK / STAT signaling genes, and upregulation of neuronal / neuroendocrine genes. Moreover, the mouse NEPC organoids show strong gene expression signatures of patient NEPC. Additionally, NEPC organoids faithfully maintain neuroendocrine features upon in vitro propagation, and developed aggressive NEPC tumors upon transplantation into mice, showing rapid tumor formation and, when transplanted orthotopically, frequent metastasis to liver and lung. Lastly, both PRAD and NEPC organoids are castration resistant prostate cancer models in that they are completely resistant to antiandrogen Enzalutamide. In summary, we have developed patient-relevant ex vivo mouse organoid systems that captured distinct states before and after in vivo PRAD-to-NEPC transformation, allowing for stringent comparison of lineage specific regulatory mechanisms.

[0216] To assess chromatin changes during PRAD-to-NEPC transition, we first performed ATAC-seq and observed a profound reprogramming of the accessible chromatin upon neuroendocrine transformation. To further identify chromatin elements and epigenetic states in NEPC and PRAD, we performed CUT&RUN profiling of six histone modifications: H3K4mel, H3K4me3, H3K36me3, H3K27ac, H3K9me3 and H3K27me3. ChromoHMM analysis using this combination of modifications revealed a dramatic epigenetic reprogramming upon neuroendocrine transformation, particularly at enhancer (active and poised) chromatin. Further analysis showed that less than 10% of NEPC enhancers are shared with PARD, as demonstrated by a profound redistribution of chromatin accessibility and enhancer modifications (H3K4mel and H3K27ac). Promoter chromatin states remained largely similar likely driven by stable expression of most housekeeping genes, which constitute the majority of genes expressed in cells. Poly comb- repressed (H3K27me3) chromatin and, to a much lesser extent, H3K9me3 heterochromatin774932-3629-0421 .1Atty. Dkt. No. 115872-3363 also show redistribution between the two states. These observations suggest enhancer reprogramming could underlie changes in gene expression upon NEPC transition.

[0217] Identification of lineage survival transcription factors in NEPC. Since TFs play fundamental roles in enhancer regulation, we hypothesized NEPC and PRAD have differential TF activity, which confers lineage-specific TF dependencies. To identify key TFs required for cell proliferation and survival in NEPC vs. PRAD, we identified 25 TFs that show strong motif enrichment at NEPC-specific (vs. PRAD) accessible chromatin and / or are highly overexpressed in NEPC. We then performed a competition based CRSIPR mini-screen by individually knocking out TF candidates in both NEPC and PRAD organoids and tracked their effect on cell fitness. Knockout of TFs critical for cell survival led to dropout of the sgRNA-containing cells from the population. The kinetics and magnitude of dropout were used to infer the dependency levels of individual targets. Using this approach, we discovered five key TF families essential for NEPC survival: proneural bHLH, MYC, FOXA, SOX and NFL Within them, seven TF members display consistent NEPC-specific / biased dependencies across both NEPC organoid lines (T70A and T101 A): Ascii, Foxal, Foxa2, Soxl, Soxl 1, Mycl and Nfib. Though expressed at similar levels in both lineages, Foxal and Nfib are more essential in NEPC, whereas the other TFs are de novo activated and exclusively required in NEPC. In addition to Mycl and Nfib, T70A showed dependency on their paralogs Mycn and Nfic. Interestingly, dependency on Myc in PRAD switches to its paralog(s) Mycl (and Mycn) upon transition to NEPC, accompanied by loss of Myc and gain of Mycl (and Mycn) expression. Since several TF paralog dependencies were observed, we next queried whether paralogs have combined functions in maintaining NEPC survival. To this end, we simultaneously knocked out Foxal / Foxa2 and Soxl / Soxl 1 in T101 A and Mycl / Mycn in T70A using dual sgRNA vectors. Double knockouts led to faster and more profound dropout in competition assays, demonstrating codepleting TF paralogs have additive effects in NEPC survival. In summary, we identified five critical and conserved families of NEPC lineage survival TFs.

[0218] Our mouse NEPC organoids model ASCL1 subtype NEPC, as demonstrated by the expression and dependency on Ascii. Human NEPC, like SCLC, has different molecular subtypes defined primarily by ASCL1 (NEPC-A) and NEURODI (NEPC-N). To assess the clinical relevance of these five families NEPC lineage survival TFs, we first examined their expression in metastatic castration-resistant prostate cancer (mCRPC) patients (NEPC vs. PRAD). Consistent with mouse organoid models, at least one member784932-3629-0421 .1Atty. Dkt. No. 115872-3363 of the proneural bHLH (ASCL1 and NEURODI), MYC (MYCL / N), FOXA (FOXA2), SOX (SOX1 / 2 / 11) families are overall upregulated in patient NEPC vs. PRAD, whereas the NFI family TFs were uniformly expressed in both lineage states (NFIB) or downregulated (NFIA / C / X). ASCL1 and NEURODI show largely mutually exclusive expression among patients. MYC and FOXA1 are overall downregulated in NEPC but remain expressed in a subset of patients.

[0219] We next functionally perturbed these five families of TFs in NEPC patient derived organoids (PDOs). We demonstrated that PDOs can be classified into two groups, NEPC-A (MSKPCal4 and LuCaP49) and NEPC-N (MSKPCalO and MSKPCa24) based on ASCL1 / NEUROD1 dependency. Notably, we showed that both NEPC-A and NEPC-N PDOs are dependent on at least one member of the proneural bHLH, MYC, FOXA and SOX families identified from the mouse system, though they display differential paralog selectivity largely driven by expression status. NEPC-A PDOs are dependent on ASCL1, MYCL (and other MYC paralogs in MSKPCal4), FOXA1, and SOX1 / SOX11, whereas NEPC-N PDOs require NEURODI, MYC, FOXA2 and SOX2. Interestingly, loss of any single NFI family TF is largely inconsequential for survival in NEPC PDOs, suggesting potential functional redundancy and compensation. Together, these results confirmed key TF families as patient-relevant lineage-specific vulnerabilities in NEPC, suggesting conserved transcriptional programs driven by oncogenic TFs are critical for lineage survival. However, they also revealed a critical challenge to target NEPC in the clinic through a single TF paralog, especially in patients with different disease subtypes or due to phenotypic heterogeneity within patient tumors, in addition to the technical challenge in pharmacologic TF targeting.

[0220] Identification of the TIP60 coactivator complex as a lineage-biased vulnerability in NEPC. To target the conserved oncogenic transcriptional programs across NEPC subtypes, we next focused on transcriptional coactivators which are essential for TF function in transcriptional regulation. To achieve precise dependency maps of potentially targetable coactivators, we performed cell competition-based CRISPR mini-screens in NEPC and PRAD organoids, focusing on 24 individual coactivator enzymes and enzymatic subunits of coactivator complexes, which represent major chromatin regulatory pathways: ATP-dependent chromatin remodeling, DNA and histone methylation, and histone acetylation. Specifically, we selected sgRNAs targeting their catalytic domains, an approach that has superior efficiency in perturbing chromatin enzymes, and knocked out either or794932-3629-0421 .1Atty. Dkt. No. 115872-3363 both paralogs when applicable to dissect potential functional redundancy. Brd4 dependency served as a positive control in both PRAD and NEPC lineages.

[0221] This screen revealed several interesting observations. (1) Several enzymes and paralog enzyme pairs displayed lineage specific / biased dependency status between NEPC and PRAD, where differential dropout kinetics was observed, confirming cell-context dependent functions of chromatin coactivators. For example, M113 / 4, Nsdl / 2, Chd7, Hbol, Tip60 and Ep400 are NEPC-biased dependencies, whereas Brgl, p300 / CBP and Gcn5 / Pcaf are more required in PRAD. (2) We observed clear functional redundancy between multiple paralogous chromatin enzymes in that single knockout had no or mild effect whereas double knockout showed strong survival defect, e.g. Brgl and Brm, M113 and M114, Nsdl and Nsd2, Ep300 and Crebbp (CBP), Gcn5 and Pcaf, Moz and Morf. (3) Histone acetylation and chromatin remodeling pathways have overall higher impact on cell survival in both lineages compared to chromatin methylation pathways.

[0222] Among the NEPC-biased vulnerabilities, both TIP60 (acetyltransferase) and p400 (ATPase / scaffold) are subunits of the mammalian TIP60 complex (also known as the NuA4 complex). In addition, Tip60 depletion leads to the fastest and most profound dropout effect in NEPC organoids. Therefore, we focused our study on the TIP60 complex as a potential target for NEPC. We next assessed the Tip60 dependency in human PRAD and NEPC PDOs. Notably, we found that Tip60 knockout led to more striking fitness defects in both NEPC subtypes (NEPC-A and NEPC-N) compared to PRAD (ARhlghand stem celllike (SCL) subtypes), demonstrating Tip60 as a neuroendocrine lineage biased yet subtype agnostic (ASCL1 vs. NEURODI) dependency. To examine the in vivo effect, we subjected NEPC organoids expressing control or Tip60 sgRNAs to subcutaneous transplantation in the mice. Loss of Tip60 strongly reduced tumor growth, demonstrating a function of Tip60 in maintaining NEPC tumor growth in vivo.

[0223] Chemical genetic targeted protein degradation reveals potential therapeutic window of TIP60 targeting in NEPC. To assess whether targeting Tip60 in NEPC has a therapeutic window, we modeled pharmacologic TIP60 perturbation in NEPC and other cellular contexts through chemical genetic targeted protein degradation. We established stable lines of mouse NEPC, PRAD and normal prostate organoids as well as immortalized dermal fibroblasts, where we first introduced a Tip60 transgene with a N-terminal FKBP12F36Vdegron tag (dTAG) at near endogenous expression levels and then knocked out the endogenous Tip60 alleles. These dTAG-Tip60 lines allowed dose dependent804932-3629-0421 .1Atty. Dkt. No. 115872-3363 degradation of TIP60 upon treatment of the VHL-recruiting degrader molecule dTAGv-l and followed largely similar degradation profile upon dTAGv-l titration. We next assessed their sensitivity to TIP60 degradation using growth / viability assays. Strikingly, we observed that NEPC displayed more than 150-, 560-, and 250-fold higher sensitivity to dTAGv-l treatment than PRAD, normal prostate and fibroblasts, respectively, demonstrating relative sensitivity in NEPC and tolerance in PRAD and normal cellular contexts to TIP60 perturbation. Parental cells without degron engineering were insensitive to dTAGv-l treatment. These observations were further confirmed using competition assays. We observed much faster dropout of the Tip60-sgRNA-expressing population in NEPC vs. other three cell types, when expressing transgenic dTAG-Tip60 and treated with 500 nM of dTAGv-l or dTAG-13 (CRBN-recruiting degrader). To further validate the higher TIP60 dependency in NEPC, we utilized CRISPRi to inhibit the transcriptional output of the endogenous Tip60 alleles at varying degrees. Consistently, we observed a higher sensitivity to Tip60 loss in NEPC compared to PRAD or normal prostate organoids. Together, these data revealed a therapeutic window for TIP60 perturbation in NEPC.

[0224] TIP60 acts as a coactivator of MYCL to integrate pro-growth transcriptional outputs of key NEPC TFs and promote lineage survival. The TIP60 complex is a potent transcriptional coactivator. We hypothesized that TIP60 regulates NEPC lineage survival transcriptional programs via acting as a coactivator of key NEPC TF(s), which underlies its sensitivity in NEPC. To this end, we first surveyed the genomic binding sites of TIP60 and dependency TFs in NEPC and PRAD. Notably, we observed a striking genomic redistribution of TIP60, especially the enhancer-bound population, from AR-occupied cis-regulatory elements (CREs) in PRAD to sites occupied by lineage survival TFs in NEPC (ASCL1, MYCL, FOXA1 / 2, SOX1 and NFIB). ASCL1, FOXA2 and SOX1 are de novo activated upon NEPC transition to occupy NEPC enhancer and promoters, whereas stably expressed FOXA1 and NFIB redistributed across the genome as TIP60. The MYC (PRAD) to MYCL (NEPC) paralog switch was also accompanied by genomic redistribution from PRAD to NEPC CREs. Of note, among the NEPC lineage survival TFs, MYCL showed highest cistrome similarity with TIP60. Next, we compared the transcriptional consequences upon short-term knockout (3 days) of TIP60 and the five families of NEPC TFs. Strikingly, gene expression changes upon Tip60 loss were highly reminiscent of that of My cl both in principal component analysis and regarding the numbers of shared differentially expressed genes. Together, these data suggested that TIP60 is814932-3629-0421 .1Atty. Dkt. No. 115872-3363 critical for activating the target genes of MYCL. TFs contain sequence-specific DNA binding activity which is absent in coactivators. We therefore queried whether MYCL directly recruits TIP60 to its target sites. We first examined whether TIP60 physically interacts with MYCL. Immunoprecipitation in the absence of DNA / RNA revealed that the TIP60 complex engages in strong protein-protein interaction with MYCL in NEPC, much more profound than with other families of TFs. To allow acute depletion of MYCL, we then established dTAG-MYCL NEPC organoids, replacing the endogenous Mycl with a dTAG- Mycl transgene. Notably, we observed a global reduction in TIP60 binding 6 hours after MYCL degradation. These findings suggested that MYCL contributes to TIP60 chromatin recruitment through physical interaction. Together, we demonstrate that TIP60 integrates into NEPC lineage survival TF network via close interplay with MYCL.

[0225] Since our data suggested that the TIP60-MYCL complex plays a critical role in NEPC lineage survival, we furthered examined the transcriptional regulation of Mycl in NEPC. Notably, Mycl expression is maintained by multiple NEPC lineage survival TFs: ASCL1, NFIB, SOX1 / 11 and MYCL itself, which all bind to its promoter suggesting direct regulation and feed-forward mechanisms. FOXA1 / 2 does not regulate Mycl expression or bind its promoter. To benchmark the transcriptional programs controlled by Mycl and Tip60 in NEPC, we showed that their knockout both led to downregulation of MYC target genes and pro-growth transcriptional programs related to E2F targets, G2M checkpoint and mTOR signaling, consistent with strong survival defects upon their depletion. Of note, these same transcriptional programs were also downregulated upon knockout of Ascii, Nfib, Soxl / 11 (but not Foxal / 2), consistent with Mycl being their downstream target. Interestingly, Tip60 or Mycl did not appear to maintain neuronal / neuroendocrine gene expression, unlike Ascii, Soxl / 11, Foxal / 2 and Nfib, indicating a designated role in regulating a subprogram in pro-growth genes. We next asked whether Mycl is the key target gene for maintaining NEPC survival downstream of ASCL1, NFIB and SOX1 / 11 by performing epistasis analysis. FOXA1 / 2 served as a negative control which promotes NEPC survival independent of MYCL. Strikingly, rescuing MYCL expression fully rescued NEPC survival upon knockout of Ascii or Nfib, whereas it was not able to rescue the Soxl / 11 loss, suggesting that Soxl / 11 regulate additional critical pro-survival expression programs. These data demonstrated that the MYCL-TIP60 complex integrates the transcriptional output of multiple NEPC TFs to promote cell cycle and proliferation transcriptional programs to promote NEPC survival.824932-3629-0421 .1Atty. Dkt. No. 115872-3363

[0226] The MYC family oncoproteins contain three paralogs, MYC, MYCN and MYCL, which display high structural similarity. Though Mycl is the key paralog expressed in mouse NEPC organoids and NEPC-A PDOs, other paralogs Myc and Mycn are expressed in substantial subsets of NEPC patients as well. Moreover, MYC is a strong dependency in NEPC-N PDOs. Therefore, we examined the interplay between other MYC paralogs with TIP60 in NEPC. We first tested the interchangeability of the three Myc paralogs in NEPC. Of note, ectopic expression of either Myc or Mycn fully rescued survival upon loss of Mycl in NEPC organoids, suggesting the three MYC paralogs are isofunctional in this context. Consistently, we observed an overall increase in MYC TF activity in NEPC patient tumors compared to PRAD. Next, we established stable NEPC organoids with knockout of endogenous Mycl and ectopic expression of dTAG-Myc or dTAG-Mycn. These organoids shifted dependency from exclusively MYCL to MYC or MYCN as demonstrated by profoundly reduced viability upon their degradation. Moreover, acute degradation of MYC or MYCN led to global decrease of TIP60 binding, reminiscent of that seen upon loss of MYCL. Together, these data demonstrated that TIP60 is a general coactivator of MYC paralogs in NEPC.

[0227] The acetyltransferase and chromatin reader activities of the TIP60 complex are required for NEPC survival. TIP60 is the acetyltransferase subunit of the mammalian TIP60 complex, a megadalton assembly of 17 or more subunits that also contains multiple chromatin binding (‘reader’) modules. We next investigated which intrinsic activities of the TIP60 complex are required to maintain NEPC survival, which may shed light on therapeutic targeting approaches. First, we examined the acetyltransferase activity of the complex. Catalytically dead Tip60 (Q377E / G380E mutant) failed to rescue NEPC survival upon knockout of endogenous Tip60, in contrast to its wild-type counterpart, demonstrating TIP60 acetyltransferase activity is essential for NEPC survival. Previous studies have shown that TIP60 can acetylate histones H4 and H2A.Z. To explore the relevant catalytic substrate(s) in NEPC, we showed that H2A.Z acetylation (H2A.Zac) was almost completely abrogated upon knockout of Tip60, whereas H4 acetylation remained largely unaltered. Furthermore, using the TIP60 degron system which allowed acute depletion, we showed that 1 hour of TIP60 degradation was sufficient for a profound loss of H2A.Zac. Total H2A.Z levels were unaffected by Tip60 knockout or degradation. Furthermore, CUT&RUN profiling and showed that H2A.Zac colocalizes with TIP60 at both active promoters and enhancers and similarly redistributes from PRAD to NEPC. Moreover, acute degradation of834932-3629-0421 .1Atty. Dkt. No. 115872-3363TIP60 led to a dramatic reduction of genomic H2A.Zac levels without affecting the deposition of H2A.Z. Together, these data demonstrated that H2A.Z is a direct and major catalytic substrate of the TIP60 acetyltransferase in NEPC. In addition, knocking out Ep400, which is the structural scaffold of the TIP60 complex and also essential for NEPC survival, led to near complete loss of H2A.Zac without affecting total H2A.Z levels in NEPC.

[0228] We then queried whether the acetylation mark itself at H2A.Z is required for NEPC survival. The histone variant H2A.Z is encoded by two genes in the mammalian genome, H2azl and H2az2. We showed that H2azl is required for NEPC survival whereas H2az2 is dispensable. Therefore, we engineered lysine-to-arginine (KR) mutants on key tail lysine residues that can be acetylated at H2A.Z1. We showed that the KR mutants lost acetylation and failed to fully rescue NEPC survival upon knockout of endogenous H2A.Z1. Interestingly, H2A.Z1 K4 / 7R (2KR) and K4 / 7 / 11 / 13 / 15R (5KR) mutants showed similar degrees of loss-of-function in this assay, suggesting K4 and K7 are predominant target residues for acetylation. These data suggested an essential role of TIP60-catalyzed H2A.Z acetylation in NEPC survival.

[0229] Next, we examined the requirement of other subunits of the TIP60 complex in NEPC survival, focusing on chromatin readers. Through systematic dependency assessment in NEPC, we found that ING3 (PHD), BRD8 (bromo) and YEATS4 (YEATS) subunits are required for NEPC survival whereas MBTD1 (MBT), MRG15 / X (chromo) are largely dispensable. Interestingly, essential subunits all contain reader domains that typically associate with promoter / enhancer-associated histone modifications, e.g. histone acetylation and H3K4me3, consistent with the genomic binding pattern of TIP60. In contrast, the reader domains of non-essential subunits MBTD1 and MRG15 / X recognize DNA damage (H4K20mel / 2) and gene body (H3K36me3) associated marks, respectively. TRRAP, a subunit that has been implicated in TF interactions, is also critical for NEPC survival. We next focused on BRD8 and YEATS4 since the associated bromodomain and YEATS domain are both canonical binding modules of histone acetylation, which broadly associates with promoters and enhancers also occupied by TIP60. Notably, knockout of Brd8 or Yeats4 led to global reduction of TIP60 occupancy, suggesting critical roles in the chromatin association of the TIP60 complex. Moreover, loss of Brd8 or Yeats4 led to a pronounced decrease of H2A.Zac, consistent with reduced TIP60 complex activity. Collectively, these findings identified key subunits of the TIP60 complex for NEPC844932-3629-0421 .1Atty. Dkt. No. 115872-3363 survival and highlighted the critical roles of acetylation-binding reader subunits in complex recruitment and cellular function.Discussion

[0230] Earlier and wider use of next generation ARSI have significantly prolonged survival of advanced PRAD patients over the past twenty years. However, the pattern of resistance to AR-targeted therapies have evolved under the strong selection pressure against this canonical prostate luminal survival pathway, in that there is an increasing percentage, now at about 20%, of patients relapse with lethal NEPC, which have undergone lineage transition to assume a neuroendocrine identity completely independent of AR activity. Similar cell lineage transitions have been observed across multiple cancers in resistance to targeted therapies.

[0231] Many TFs have been studies individually in NEPC but a systematic analysis was lacking. We identified five families of TFs that are consistently critical for NEPC, revealed paralog selectivity between NEPC-A and NEPC-N, shown that MYCL integrates the transcriptional output of multiple NEPC TFs to control pro-proliferation oncogenic programs using TIP60 as a key coactivator., and shown that ASCL1 regulates both key neuronal / neuroendocrine lineage transcriptional program and MYCL-driven proliferation program. We further established that the TIP60 complex proteins BRD8 and YEATS4 are also dependencies, likely though reinforcing chromatin binding through their respective BRD domains following recruitment by LMYC. We further established the therapeutic use of inhibitors of the TIP60, including catalytic acetyltransferase inhibitors, as TIP60 activity is important to NEPC survival.

[0232] The results described herein establish TIP60’s importance to the transition to and maintenance of the NEPC cell state and the therapeutic application of TIP60 inhibitors to prevent or reverse said transition. Accordingly, the methods of the present disclosure are effective for treating prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a TIP60 inhibitor and an effective amount of an agent that specifically targets prostate adenocarcinoma (PRAD) lineage cells or lung adenocarcinoma (LU D) lineage cells in tumors.EQUIVALENTS

[0233] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of854932-3629-0421 .1Atty. Dkt. No. 115872-3363 individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0234] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0235] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

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Claims

Atty. Dkt. No. 115872-3363CLAIMS1. A method for treating or preventing prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a LATS kinase inhibitor.

2. A method for enhancing responsiveness of a prostate cancer patient or a lung cancer patient to Androgen Receptor Signaling Inhibitor (ARSI) comprising sequentially, simultaneously or separately administering to the patient an effective amount of a LATS kinase inhibitor and an effective amount of an ARSI.

3. The method of claim 2, wherein the ARSI is selected from the group consisting of abiraterone acetate, apalutamide, bicalutamide, clascoterone, enzalutamide, flutamide, ARN-509, nilutamide and darolutamide.

4. A method for treating prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a LATS kinase inhibitor and an effective amount of an agent that specifically targets prostate adenocarcinoma (PRAD) lineage cells or lung adenocarcinoma (LU AD) lineage cells in tumors.

5. The method of claim 4, wherein the agent that specifically targets PRAD or LU AD lineage cells binds to STEAP1 or PSMA.

6. The method of claim 5, wherein the agent that specifically targets PRAD or LU AD lineage cells is an antibody drug conjugate, a T cell engager, a CAR T cell or a radioligand, optionally wherein the radioligand is lutetium- 177-PSMA-617.

7. The method of any one of claims 1-6, wherein the LATS kinase is LATS1 or LATS2.

8. The method of any one of claims 1-7, wherein the LATS kinase inhibitor is selected from among: TDI-011536, VT02956, GA-017, NIBR, 4-(4-(4-(4-Chlorophenyl)piperidin- 4-yl)phenyl)-lH-pyrrolo[2,3-b]pyridine, 4-(4-(Piperidin-4-yl)phenyl)-lH-pyrrolo[2,3- b]pyridine, 3-Fluoro-4-(4-(piperidin-4-yl)phenyl)-lH-pyrrolo[2,3-b]pyridine, 4-(4-(3- Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)piperidin-4-ol, 3 -(4-(3 -Fluoro- 1H- pyrrolo[2,3-b]pyridin-4-yl)phenyl)piperidin-3-ol, 3-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin- 4-yl)phenyl)pyrrolidin-3-ol, 3-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)azetidin- 3-ol, 4-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)azepan-4-ol, (lR,5S)-3-(4-(3- Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol, 6-(4-(3-914932-3629-0421 .1Atty. Dkt. No. 115872-3363Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-2-azaspiro[3.3]heptan-6-ol, 4-(4-(3-Fluoro- lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3-methylpiperidin-4-ol, (3R,4s,5S)-4-(4-(3-Fluoro- lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)-4-(2-Fluoro- 4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)- 4-(2-Chloro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-3,5-dimethylpiperidin-4-ol, (3S,4s,5R)-4-(4-(3-Fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-2-methylphenyl)-3,5- dimethylpiperidin-4-ol, (3S,4s,5R)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)- 6-methylphenyl)-3,5-dimethylpiperidin-4-ol, (3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH- pyrrolo[2,3-b]pyridin-4-yl)-6-methylphenyl)-l,3,5-trimethylpiperidin-4-ol, (3R,4s,5S)-4-(2- Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)-6-methylphenyl)-l-isopropyl-3,5- dimethylpiperidin-4-ol, (3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3-b]pyridin-4-yl)- 6-methylphenyl)-3,5-dimethyl-l-(oxetan-3-yl)piperidin-4-ol, l-((3R,4s,5S)-4-(2-Fluoro-4- (3 -fluoro- 1 H-pyrrolo [2, 3 -b]pyridin-4-yl)-6-methylphenyl)-4-hy droxy-3 , 5 - dimethylpiperidin-l-yl)ethan-l-one, 4-((3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-lH-pyrrolo[2,3- b]pyridin-4-yl)-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-l-yl)tetrahydro-2H- thiopyran 1,1 -di oxide,924932-3629-0421 .1Atty. Dkt. No. 115872-33639. A method for treating or preventing prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a TIP60 inhibitor.

10. A method for enhancing responsiveness of a prostate cancer patient or a lung cancer patient to Androgen Receptor Signaling Inhibitor (ARSI) comprising sequentially, simultaneously or separately administering to the patient an effective amount of a TIP60 inhibitor and an effective amount of an ARSI.

11. The method of claim 10, wherein the ARSI is selected from the group consisting of abiraterone acetate, apalutamide, bicalutamide, clascoterone, enzalutamide, flutamide, ARN-509, nilutamide and darolutamide.

12. A method for treating prostate cancer or lung cancer in a patient in need thereof comprising administering to the patient an effective amount of a TIP60 inhibitor and an effective amount of an agent that specifically targets prostate adenocarcinoma (PRAD) lineage cells or lung adenocarcinoma (LU AD) lineage cells in tumors.

13. The method of claim 12, wherein the agent that specifically targets PRAD or LU AD lineage cells binds to STEAP1 or PSMA.

14. The method of claim 13, wherein the agent that specifically targets PRAD or LUAD lineage cells is an antibody drug conjugate, a T cell engager, a CAR T cell or a radioligand, optionally wherein the radioligand is lutetium- 177-PSMA-617.

15. The method of any one of claims 9-14, wherein the TIP60 inhibitor is selected from among TH1834, NU9056, Pentamidine, Acetyl CoA, and MG149.

16. The method of any one of claims 1-15, wherein the prostate cancer or lung cancer harbors a mutation in Rbl and / or Trp53.

17. The method of any one of claims 1-16, wherein the prostate cancer or lung cancer comprise ASCL1+ cells.934932-3629-0421 .1Atty. Dkt. No. 115872-336318. The method of any one of claims 1-17, wherein the prostate cancer is neuroendocrine prostate cancer (NEPC) or the lung cancer is lung neuroendocrine cancer.944932-3629-0421 .1

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