Method of treating prostate cancer with dual pathway regulation of castration-response in the prostate epithelium

Abstract

The subject matter described herein relates to a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising modulating the expression of a first component of an intrinsic androgen signaling pathway and modulating the expression of a second component of an extrinsic androgen signaling pathway.

Description

Attorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025METHOD OF TREATING PROSTATE CANCER WITH DUAL PATHWAY REGULATION OF CASTRATION-RESPONSE IN THE PROSTATE EPITHELIUM

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 696,344, filed on September 18, 2024, the content of which is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

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

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

[0004] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosure of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described herein.SUMMARY OF THE INVENTION

[0005] In certain aspects, the subject matter described herein provides a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising modulating the expression of a first component of an intrinsic androgen signaling pathway. In some embodiments, the method further comprises modulating the expression of a second component of an extrinsic androgen signaling pathway. In some embodiments, the prostate cancer is an early-stage prostate cancer, a Stage 0 prostate cancer, a Stage I prostate cancer, a Stage II prostate cancer, a Stage III prostate cancer, a Stage IV prostate cancer, an advanced stage, or an end-stage cancer. In some embodiments, the prostate cancer is castrationresistant prostate cancer (CRPC), neuroendocrine prostate cancer (NEPC), neuroendocrine castration resistant prostate cancer (CRPC-NE), CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0006] In some embodiments, the extrinsic androgen signaling pathway occurs at least partially in a stromal microenvironment. In some embodiments, the extrinsic androgen signaling pathway occurs partially in a stromal microenvironment and partially within a1ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 prostate luminal epithelial cell. In some embodiments, the intrinsic androgen signaling pathway occurs at least partially within a prostate luminal epithelial cell. In some embodiments, the intrinsic androgen signaling pathway occurs within a prostate luminal epithelial cell.

[0007] In some embodiments, the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises inducing ferroptosis by downregulating or inhibiting the intrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises inducing lipid peroxidation by downregulating or inhibiting the intrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises disrupting the balance of Monounsaturated Fatty Acids / Polyunsaturated Fatty Acids (MUFA / PUFA) by downregulating or inhibiting the intrinsic androgen signaling pathway.

[0008] In some embodiments, the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises inducing ferroptosis by downregulating or inhibiting the extrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises inducing lipid peroxidation by downregulating or inhibiting the extrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises disrupting the balance of Monounsaturated Fatty Acids / Polyunsaturated Fatty Acids (MUFA / PUFA) by downregulating or inhibiting the extrinsic androgen signaling pathway.

[0009] In some embodiments, wherein the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises inhibiting ferroptosis by upregulating or activating the intrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises inhibiting lipid peroxidation by upregulating or activating the intrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises maintaining the balance of Monounsaturated Fatty Acids / Polyunsaturated Fatty Acids (MUFA / PUFA) by upregulating or activating the intrinsic androgen signaling pathway. In some embodiments, the intrinsic androgen signaling pathway is upregulated or activated in a noncancerous cell.

[0010] In some embodiments, wherein the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises inhibiting ferroptosis by2ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 upregulating or activating the extrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises inhibiting lipid peroxidation by upregulating or activating the extrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises maintaining the balance of Monounsaturated Fatty Acids / Polyunsaturated Fatty Acids (MUFA / PUFA) by upregulating or activating the extrinsic androgen signaling pathway. In some embodiments, the intrinsic androgen signaling pathway is upregulated or activated in a noncancerous cell.

[0011] In some embodiments, the first component is NKX3.1, FASN, ACSL3, MBOAT2, or GPX4. In some embodiments, the second component is pleiotrophin (PTN), mTORCl, SREBP1, or SCD1.

[0012] In certain aspects, the subject matter described herein provides a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a first inhibitor and a therapeutically effective amount of a second inhibitor, wherein the first inhibitor modulates the expression of a component of an intrinsic androgen signaling pathway and the second inhibitor modulates the expression of a component of an extrinsic androgen signaling pathway. In some embodiments, the prostate cancer is an early-stage prostate cancer, a Stage 0 prostate cancer, a Stage I prostate cancer, a Stage II prostate cancer, a Stage III prostate cancer, a Stage IV prostate cancer, an advanced stage, or an end-stage cancer. In some embodiments, the prostate cancer is castrationresistant prostate cancer (CRPC), neuroendocrine prostate cancer (NEPC), neuroendocrine castration resistant prostate cancer (CRPC-NE), CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0013] In some embodiments, the extrinsic androgen signaling pathway occurs at least partially in a stromal microenvironment. In some embodiments, the extrinsic androgen signaling pathway occurs partially in a stromal microenvironment and partially within a prostate luminal epithelial cell. In some embodiments, the intrinsic androgen signaling pathway occurs at least partially within a prostate luminal epithelial cell. In some embodiments, the intrinsic androgen signaling pathway occurs within a prostate luminal epithelial cell.3ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025

[0014] In some embodiments, the first inhibitor comprises a NKX3.1 inhibitor, a FASN inhibitor, a ACSL3 inhibitor, a MBOAT2 inhibitor, GPX4 inhibitor, or any combination thereof.

[0015] In some embodiments, the first inhibitor comprises a composition comprising an inhibitory RNA for a messenger RNA sequence encoding a NKX3.1 protein, an inhibitory RNA for a messenger RNA sequence encoding a FASN protein, an inhibitory RNA for a messenger RNA sequence encoding a ACSL3 protein, an inhibitory RNA for a messenger RNA sequence encoding a MBOAT2 protein, an inhibitory RNA for a messenger RNA sequence encoding a GPX4 protein, or any combination thereof.

[0016] In some embodiments, the first inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a NKX3.1 gene or controlling transcription of a NKX3.1 gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a FASN gene or controlling transcription of a FASN gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding aACSL3 gene or controlling transcription of aACSL3 gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding aMBOAT2 gene or controlling transcription of aMBOAT2 gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a GPX4 gene or controlling transcription of a GPX4 gene, or any combination thereof.

[0017] In some embodiments, the second inhibitor comprises a composition comprising an inhibitory RNA for a messenger RNA sequence encoding a PTN protein, an inhibitory RNA for a messenger RNA sequence encoding a MTOR protein, an inhibitory RNA for a messenger RNA sequence encoding a mTORCl protein, an inhibitory RNA for a messenger RNA sequence encoding a RPTOR protein, an inhibitory RNA for a messenger RNA sequence encoding a SREBP1 protein, an inhibitory RNA for a messenger RNA sequence encoding a SCD1 protein, or any combination thereof.

[0018] In some embodiments, the second inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a PTN gene or controlling transcription of a PTN gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide sequence encoding a MTOR gene or controlling transcription of the MTOR gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a mTORCl gene or controlling transcription of a mTORCl gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide sequence encoding a RPTOR gene or controlling transcription of the RPTOR gene, a CRISPR4ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a SREBP1 gene or controlling transcription of a SREBP1 gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a SCD1 gene or controlling transcription of a SCD1 gene, or any combination thereof.

[0019] In some embodiments, the first inhibitor comprises a NKX3.1 inhibitor, a FASN inhibitor, a ACSL3 inhibitor, a MBOAT2 inhibitor, GPX4 inhibitor, or any combination thereof and the second inhibitor comprises a PTN inhibitor, a MTOR inhibitor, a mTORCl inhibitor, a SREBP1 inhibitor, a SCD1 inhibitor, or any combination thereof. In some embodiments, the first inhibitor comprises a composition comprising an inhibitory RNA for a messenger RNA sequence encoding a NKX3.1 protein and wherein the second inhibitor comprises a composition comprising an inhibitory RNA for a messenger RNA sequence encoding a PTN protein. In some embodiments, the first inhibitor comprises a NKX3.1 inhibitor and wherein the second inhibitor comprises a PTN inhibitor.

[0020] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment. In some embodiments, the antiandrogen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide. In some embodiments, the antiandrogen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists. In some embodiments, wherein the pharmaceutical composition is administered before, after, or in combination with radiation therapy. In some embodiments, the administration of the pharmaceutical composition to the subject in combination with an anti-androgen treatment decreases tumor size. In some embodiments, the administration of the pharmaceutical composition to the subject in combination with an anti-androgen treatment decreases tumor number. In some embodiments, the administration of the pharmaceutical composition to the subject in combination with an anti-androgen treatment prevents cancer metastasis. In some embodiments, the subject is human.BRIEF DESCRIPTION OF FIGURES

[0021] The patent or application file contains at least one drawing originally in color. To conform to the requirements for PCT patent applications, many of the figures presented herein are black and white representations of images originally created in color.

[0022] FIGS. 1A-E shows how loss of stromal-epithelial interactions mediates castration-induced prostate regression. FIGS. 1A-C show a conceptual model for androgen receptor (AR) function in stroma and epithelium. (A) Stromal AR promotes survival of5ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 epithelial cells through production of an “anti-death” signal. Luminal AR also promotes a survival signal. (B) Following androgen-deprivation, the loss of both stromal and luminal AR anti-death signals leads to cell death and prostate regression. (C) In the castration-resistant state, activation of the Akt-mTOR pathway, among others, can bypass AR to promote antideath signaling (see Ref. 2). FIG. ID illustrates the main components in the prostate (see Ref. 2). FIG. IE are dissected mice anterior prostate (AP) lobes after castration and when testosterone is subsequently administered (see Ref. 2).

[0023] FIGS. 2A-C provide illustrations of ferroptosis. FIG. 2A provides an overview of the ferroptosis pathway (see Ref. 97). FIG. 2B shows the markers of ferroptosis (see Ref. 22). FIG. 2C provides a detailed illustration of the dual pathway mechanism for induction of ferroptosis following androgen-deprivation.

[0024] FIGS. 3A-C shows the epithelial heterogeneity and proximal-distal asymmetry in the mouse prostate. FIG. 3A is an aggregated composite plot of published and new scRNA- seq datasets for mouse prostate (see Ref 31). FIG. 3B is a dot plot of gene expression levels in each epithelial population for selected markers (see Ref 24). FIG. 3C is the schematic model of prostate lobes indicating the distribution of luminal epithelial populations (see Ref 24). FIG. 3D is a sharp boundary of gene expression divides proximal from distal regions.

[0025] FIGS. 4A-G show defining features of prostate regression following castration. FIG. 4A shows the dark-field and immunofluorescence (IF) staining of dissected anterior prostate (AP) lobes from wild type (WT) C57BL / 6 mice that were hormonally intact or castrated for the indicated number of days. FIGS. 4B-C show plots of prostate lobe weight (FIG. B) and luminal cell size (FIG. C) in AP lobes from intact and castrated WT mice. FIG. 4D are plots of cell size using forward scatter area (FSC-A) of sorted distal and proximal YFP-positive luminal cells from tamoxifen-treated Nkx3.1CreERT2 / +; R26R-YFP mice (NY), which labels distal luminal cells, and Krt7CreERT2 / +; R26R-YFP mice (KY), which labels proximal luminal cells. FIG. 4E shows a plot of luminal cell number in AP lobes from intact and castrated WT mice. FIG. 4F are scatterplots of scRNA-seq data for AP luminal cells from hormonally intact or castrated mice. FIG. 4G is the schematic of the experimental analysis.

[0026] FIGS. 5A-H show that luminal cell death is mediated by ferroptosis. FIG. 5A provides the luminal cell numbers in AP lobes at day 28 after castration after blocking different cell death pathways. FIG. 5B is the lipidomic analysis of the most abundant polyunsaturated fatty acids (PUFAs) in luminal cells from intact and castrated WT mice. FIG. 5C shows the immunoblotting of proteins involved in mTORCl activity and MUFA synthesis in6ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 AP luminal cells from WT and castrated mice, using samples from three biological replicates. FIG. 5D are scanning electron micrographs of distal luminal cells. FIGS. 5E-F are lots of mitochondrial cristae number and area, respectively. FIG. 5G shows the experimental strategy for testing the in vivo roles of ferroptosis, apoptosis, and pyroptosis in luminal cell death after castration. FIG. 5H is the lobe weight and luminal cell number in control and liproxstatin-1 treated mice at Cas d28.

[0027] FIGS. 6A-G show that androgen receptor has distinct roles in stroma and epithelium. FIGS. 6A, F are dark-field and IF images of dissected AP lobes from mice with induced deletion of Ar in luminal cells (Lum AR KO), stromal cells (Str AR KO), or both (Lum + Str AR KO), at day 28 after tamoxifen induction (see text for genotypes). FIGs. 6B- D, G are plots of AP lobe weight (FIG. 6B), distal luminal cell size (FIG. 6C), proximal luminal cell size (FIG. 6G), and luminal cell number (FIG. 6D) from mice with deletion of Ar in luminal cells, stromal cells, or both at day 28 after tamoxifen induction. FIG. 6E show transcriptomic changes in AP luminal cells following Ar deletion in luminal cells, stromal cells, or both at day 28 after tamoxifen induction.

[0028] FIGS. 7A-M shows that intrinsic signaling is mediated by activities of NKX3.1. FIGS. 7A-B show AP lobe weight (FIG. 7A) and luminal cell number (FIG. 7B) from mice with homozygous null Nkx3.1 mutation and / or stromal Ar deletion at day 28 days after tamoxifen induction. FIGS. 7C-D are measurements of GSH (FIG. 7C) and GSH / GSSG ratio (FIG. 7D) in AP luminal cells from WT and Nkx3.1 mutant mice. FIG. 7E are immunoblotting of proteins involved in mono-unsaturated fatty acid (MUFA) synthesis in AP luminal cells isolated from WT and Nkx3.1 mutant mice, using samples from three biological replicates. FIGS. 7F-G show the abundance of representative MUFA-phospholipids in luminal cells from WT intact and Cas d7 mice (FIG. 7F) and from mice with induced deletion of Ar at day 7 after tamoxifen induction (FIG. 7G), with each dot corresponding to lipids extracted from combined luminal cells from 5 mice. FIG. 7H shows AR and NKX3.1 ChlP-seq signal tracks near Gpx4 and MUFA synthesis gene loci in mouse prostate datasets. FIGS. I-J are dark-field images of AP lobes and immunofluorescence staining of AP sections from control mice, and mice with Nkx3.1 mutant (Nkx3.1 KO), induced deletion of Ar in stromal cells (Str Ar KO), or both (Str Ar + Nkx3.1 KO), at day 28 after tamoxifen induction. Scale bars for whole-mounts, 1 mm; for ections, 20 pm. FIGS. 7K-L are luminal cell sizes in mice of the indicated genotypes at day 28 after tamoxifen induction. FIG. 7M are scatterplots of scRNA-seq data showing transcriptomic changes in distal (blue) and proximal (red) luminal cells from control mice or Nkx3.1 mutant mice.7ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025

[0029] FIGS. 8A-K shows that extrinsic signaling utilizes a PTN-mTORCl-SCDl axis. FIGS. 8A-B show AP lobe weight (FIG. 8A) and luminal cell number (FIG. 8B) from mice with stromal deletion of Ptn and / or Nkx3.1 homozygous mutation at 28 days after tamoxifen induction. FIG. 8C are the immunoblottings of proteins involved in mTORCl activity and MUFA synthesis in luminal cells from WT and stromal deleted Ptn mice, using samples from three biological replicates. FIGS. 8D-E show the AP lobe weights (FIG. 8D) and luminal cell numbers (FIG. 8E) from mice with luminal deletion of Raptor and / or Ar at 28 days after tamoxifen induction; p-values were calculated by t tests. FIG. 8F are the immunoblottings of proteins involved in mTORCl activity and MUFA synthesis in WT and Raptor deleted luminal cells, using samples from three biological replicates. FIG. 8G show dark-field images of AP lobes and immunofluorescence staining of AP sections from control mice and mice with induced deletion of Ar in luminal cells (Lum Ar KO), Raptor in luminal cells (Lum Raptor KO), or both (Lum Raptor + Ar KO), at day 28 after tamoxifen induction. FIG. 8H is a volcano plot displaying significantly altered prostate stromal ligands from RNA-seq analyses of stromal cells from WT-intact and 3-day castrated mice. FIG. 81 is the western blotting of IGF1R and mTORCl pathway proteins in luminal cells from the indicated genotypes at 7 days after tamoxifen treatment. FIGS. 8J-K are plots of AP lobe weight and luminal cell number from mice of the indicated genotypes at 28 days after castration.

[0030] FIGS. 9A-B shows evidence for ferroptosis in human prostate after ADT or enzalutamide treatment. FIG. 9A is the immunohistochemical (IHC) staining for AMACR, a marker of prostate tumors, and TFR1, a marker of ferroptosis, in benign prostate and prostate tumors from treatment-naive patients or patients who received neoadjuvant ADT prior to transurethral resection or prostatectomy. FIG. 9B is the gene set enrichment analysis (GSEA) using bulk RNA-seq data from tumor biopsy samples from mCRPC patients who were responders or non-responders to enzalutamide treatment (see Ref 54).

[0031] FIG. 10 shows the multiome analysis of chromatin accessibility in prostate epithelial cells.

[0032] FIGS. 11A-D show the time course of luminal cell proliferation and apoptosis in wild type mouse prostate after castration. FIG. 11 A show the staining of DAPI, Ki67, luminal cell specific marker CK8, terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL) in prostate tissue sections after castration. FIGS. 11B-C are the quantifications of cell proliferation (FIG. 11B) or apoptosis (FIG. 11C) in luminal cells from WT mice FIG. 11D are ridgeplots for the distribution of gene set enrichment (GSEA)8ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 results for four cell death signatures in luminal cells, comparing scRNA-seq data for WT Cas d7 versus WT intact prostate.

[0033] FIGS. 12A-B relate to the generation of stromal-specific Ar deletion mouse model. FIG. 12A provides the experimental strategy and mouse genotypes for inducible deletion of AR in prostate luminal cells and / or stromal cells. FIG. 12B is the immunostaining for AR, CK5, CK8, and Vimentin (VIM) in the AP lobes of the indicated genotypes to delete AR in luminal cells, stromal cells, or both at 28 days after tamoxifen- induction.BACKGROUND

[0034] Prostate cancer is one of the most common types of cancer in men. It is known that deprivation of male hormones (androgens) results in profound shrinkage of normal prostate (FIG. IE) as well as hormone-sensitive prostate tumors. Consequently, androgendeprivation therapy (ADT) has represented first-line therapy for prostate cancer for decades, and development of potent anti-androgens has resulted in successful therapies for tumors that ultimately recur following ADT. Despite these decades of clinical advances, however, the basis for castration-sensitivity has remained poorly understood.

[0035] The prostate mainly contains luminal, basal and rara neuroendocrine epithelial cells as well as the surrounding stromal cells (FIG. ID). Luminal epithelial cells make up the most part of the prostate. Both luminal, basal and stromal cells are AR positive. In the past several decades, it was believed that extrinsic androgen signaling in the stromal cells maintains prostate homeostasis through stromal-epithelial interactions while intrinsic luminal androgen signaling is dispensable for lumina cell survival. Apoptosis was believed to be the major type of cell death for castration-induced prostate regression.DETAILED DESCRIPTION OF THE INVENTION

[0036] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0037] As would be apparent to one of ordinary skill in the art, any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0038] These, and other, embodiments of the invention will be better appreciated and understood when considered in conjunction with the following description and the9ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 accompanying drawings. It should be understood, however, that the following description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions and / or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and / or rearrangements.

[0039] An “effective amount”, “sufficient amount” or “therapeutically effective amount” as used herein is an amount of a compound that is sufficient to effect beneficial or desired results, including clinical results. As such, the effective amount may be sufficient, for example, to reduce or ameliorate the severity and / or duration of an affliction or condition, or one or more symptoms thereof, prevent the advancement of conditions related to an affliction or condition, prevent the recurrence, development, or onset of one or more symptoms associated with an affliction or condition, or enhance or otherwise improve the prophylactic or therapeutic effect(s) of another therapy. An effective amount also includes the amount of the compound that avoids or substantially attenuates undesirable side effects.

[0040] The terms “animal,” “subject” and “patient” as used herein includes all members of the animal kingdom including, but not limited to, mammals, animals (e.g., cats, dogs, horses, swine, etc.) and humans.

[0041] The corresponding NCBI Gene IDs for the genes are provided herein. The full nucleic acid and amino acid sequences of these genes can be readily obtained by accessing the National Center for Biotechnology Information (NCBI) database using the provided Gene IDs. These sequences are publicly available and can be retrieved using standard bioinformatics tools or direct database queries. The nucleic acid and amino acid sequences of the genes identified by their respective Gene IDs are hereby incorporated by reference in their entirety.

[0042] The corresponding PubChem ID for the compounds are provided herein. The information of the compound can be readily obtained by accessing the National Center for Biotechnology Information (NCBI) PubChem database using the provided PubChem IDs. The information are publicly available and are hereby incorporated by reference in their entirety.

[0043] In one aspect, provided herein is a method of treating or preventing prostate cancer in a subject in need thereof, comprising modulating the expression of a first component of an intrinsic androgen signaling pathway.10ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025

[0044] In some embodiments, the method further comprises modulating the expression of a second component of an extrinsic androgen signaling pathway.

[0045] In some embodiments, the prostate cancer is Stage 0 prostate cancer. In some embodiments, the prostate cancer is Stage I prostate cancer. In some embodiments, the prostate cancer is Stage II prostate cancer. In some embodiments, the prostate cancer is Stage III prostate cancer. In some embodiments, the prostate cancer is Stage IV prostate cancer. In some embodiments, the prostate cancer is early-stage prostate cancer. In some embodiments, the prostate cancer is an advanced stage or an end-stage cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is a neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE). In some embodiments, the prostate cancer is CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0046] In some embodiments, the extrinsic androgen signaling pathway occurs at least partially in a stromal microenvironment. In some embodiments, the extrinsic androgen signaling pathway occurs partially in a stromal microenvironment and partially within a prostate luminal epithelial cell. In some embodiments, the intrinsic androgen signaling pathway occurs at least partially within a prostate luminal epithelial cell. In some embodiments, the intrinsic androgen signaling pathway occurs within a prostate luminal epithelial cell.

[0047] In some embodiments, the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises inducing ferroptosis by downregulating or inhibiting the intrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises inducing lipid peroxidation by downregulating or inhibiting the intrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises disrupting the balance of Monounsaturated Fatty Acids / Polyunsaturated Fatty Acids (MUFA / PUFA) by downregulating or inhibiting the intrinsic androgen signaling pathway.

[0048] In some embodiments, the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises inducing ferroptosis by downregulating or inhibiting the extrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises11ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 inducing lipid peroxidation by downregulating or inhibiting the extrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises disrupting the balance of Monounsaturated Fatty Acids / Polyunsaturated Fatty Acids (MUFA / PUFA) by downregulating or inhibiting the extrinsic androgen signaling pathway.

[0049] In some embodiments, modulating the expression of a first component of an intrinsic androgen signaling pathway comprises inhibiting ferroptosis by upregulating or activating the intrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises inhibiting lipid peroxidation by upregulating or activating the intrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises maintaining the balance of Monounsaturated Fatty Acids / Polyunsaturated Fatty Acids (MUFA / PUFA) by upregulating or activating the intrinsic androgen signaling pathway. In some embodiments, the intrinsic androgen signaling pathway is upregulated or activated in a noncancerous cell.

[0050] In some embodiments, the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises inhibiting ferroptosis by upregulating or activating the extrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises inhibiting lipid peroxidation by upregulating or activating the extrinsic androgen signaling pathway. In some embodiments, the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises maintaining the balance of Monounsaturated Fatty Acids / Polyunsaturated Fatty Acids (MUFA / PUFA) by upregulating or activating the extrinsic androgen signaling pathway. In some embodiments, the intrinsic androgen signaling pathway is upregulated or activated in a noncancerous cell.

[0051] In some embodiments, the first component is NKX3.1, FASN, ACSL3, MBOAT2, or GPX4. In some embodiments, the second component is pleiotrophin (PTN), MTOR, mTORCl, SREBP1, or SCD1.

[0052] In another aspect, provided herein is a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a NKX3.1 inhibitor.

[0053] In some embodiments, the prostate cancer is Stage 0 prostate cancer. In some embodiments, the prostate cancer is Stage I prostate cancer. In some embodiments, the12ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 prostate cancer is Stage II prostate cancer. In some embodiments, the prostate cancer is Stage III prostate cancer. In some embodiments, the prostate cancer is Stage IV prostate cancer. In some embodiments, the prostate cancer is early-stage prostate cancer. In some embodiments, the prostate cancer is an advanced stage or an end-stage cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is a neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE). In some embodiments, the prostate cancer is CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0054] In some embodiments, the NKX3.1 inhibitor is administered in combination with another NKX3.1 inhibitor or one or more NKX3.1 activity modulator.

[0055] In some embodiments, the NKX3.1 inhibitor comprises a composition comprising a small interfering RNA specific for a messenger RNA sequence encoding a NKX3.1 protein. In some embodiments, the inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding NKX3.1 gene (NCBI Gene ID: 4824) or controlling transcription of NKX3.1 gene.

[0056] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment.

[0057] In some embodiments, the anti-androgen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide.

[0058] In some embodiments, the anti-androgen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

[0059] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with radiation therapy.

[0060] In some embodiments, the administration of the NKX3.1 inhibitor to the subject in combination with an anti-androgen treatment decreases tumor size. In some embodiments, the administration of the NKX3.1 inhibitor to the subject in combination with an antiandrogen treatment decreases tumor number. In some embodiments, the administration of the NKX3.1 inhibitor to the subject in combination with an anti-androgen treatment prevents cancer metastasis.

[0061] In another aspect, provided herein is a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to the subject a13ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 pharmaceutical composition comprising a therapeutically effective amount of a PTN inhibitor.

[0062] In some embodiments, the prostate cancer is Stage 0 prostate cancer. In some embodiments, the prostate cancer is Stage I prostate cancer. In some embodiments, the prostate cancer is Stage II prostate cancer. In some embodiments, the prostate cancer is Stage III prostate cancer. In some embodiments, the prostate cancer is Stage IV prostate cancer. In some embodiments, the prostate cancer is early-stage prostate cancer. In some embodiments, the prostate cancer is an advanced stage or an end-stage cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC).

[0063] In some embodiments, the prostate cancer is a neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE). In some embodiments, the prostate cancer is CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0064] In some embodiments, the PTN inhibitor is administered in combination with another PTN inhibitor or one or more PTN activity modulator.

[0065] In some embodiments, the PTN inhibitor comprises a composition comprising a small interfering RNA specific for a messenger RNA sequence encoding a PTN protein. In some embodiments, the inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a. PTN gene (NCBI Gene ID: 5764) or controlling transcription of a PTN gene.

[0066] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment.

[0067] In some embodiments, the anti-androgen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide.

[0068] In some embodiments, the anti-androgen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

[0069] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with radiation therapy.

[0070] In some embodiments, the administration of the PTN inhibitor to the subject in combination with an anti-androgen treatment decreases tumor size. In some embodiments, the administration of the PTN inhibitor to the subject in combination with an anti-androgen treatment decreases tumor number. In some embodiments, the administration of the PTN14ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 inhibitor to the subject in combination with an anti-androgen treatment prevents cancer metastasis.

[0071] In another aspect, provided herein is a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a MTOR inhibitor.

[0072] In another aspect, provided herein is a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a mTORCl inhibitor.

[0073] In some embodiments, the MTOR inhibitor is administered in combination with another MTOR inhibitor or one or more MTOR activity modulators.

[0074] In some embodiments, the prostate cancer is Stage 0 prostate cancer. In some embodiments, the prostate cancer is Stage I prostate cancer. In some embodiments, the prostate cancer is Stage II prostate cancer. In some embodiments, the prostate cancer is Stage III prostate cancer. In some embodiments, the prostate cancer is Stage IV prostate cancer. In some embodiments, the prostate cancer is early-stage prostate cancer. In some embodiments, the prostate cancer is an advanced stage or an end-stage cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is a neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE). In some embodiments, the prostate cancer is CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0075] In some embodiments, the mTORCl inhibitor is administered in combination with another mTORCl inhibitor or one or more mTORCl activity modulator.

[0076] In some embodiments, the mTORCl inhibitor comprises a composition comprising a small interfering RNA specific for a messenger RNA sequence encoding the mTOR protein. In some embodiments, the inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a. MTOR gene or controlling transcription of MTOR gene. The NCBI Gene ID of the MTOR gene, which encodes the mTOR subunit of mTORCl and mTORC2, is 2475.

[0077] In some embodiments, the mTORCl inhibitor targets one or more components of the mTORCl complex, e.g. MTOR, Raptor. In some embodiments, the mTORCl inhibitor15ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 targets an mTORCl component, such as Raptor (Regulatory Associated Protein of MTOR Complex 1). In some embodiments, the mTORCl inhibitor comprises a composition comprising a small interfering RNA (siRNA) specific for a messenger RNA sequence encoding RPTOR. In some embodiments, the mTORCl inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide sequence encoding a RPTOR gene (NCBI Gene ID: 57521) or controlling transcription of the RPTOR gene.

[0078] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment.

[0079] In some embodiments, the anti-androgen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide.

[0080] In some embodiments, the anti-androgen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

[0081] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with radiation therapy.

[0082] In some embodiments, the administration of the mTORCl inhibitor to the subject in combination with an anti-androgen treatment decreases tumor size. In some embodiments, the administration of the mTORCl inhibitor to the subject in combination with an antiandrogen treatment decreases tumor number. In some embodiments, the administration of the mTORCl inhibitor to the subject in combination with an anti-androgen treatment prevents cancer metastasis.

[0083] In some embodiments, the administration of the MTOR inhibitor to the subject in combination with an anti-androgen treatment decreases tumor size. In some embodiments, the administration of the MTOR inhibitor to the subject in combination with an anti-androgen treatment decreases tumor number. In some embodiments, the administration of the MTOR inhibitor to the subject in combination with an anti-androgen treatment prevents cancer metastasis.

[0084] In another aspect, provided herein is a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a SREBP1 inhibitor.

[0085] In some embodiments, the prostate cancer is Stage 0 prostate cancer. In some embodiments, the prostate cancer is Stage I prostate cancer. In some embodiments, the prostate cancer is Stage II prostate cancer. In some embodiments, the prostate cancer is Stage III prostate cancer. In some embodiments, the prostate cancer is Stage IV prostate cancer. In16ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 some embodiments, the prostate cancer is early-stage prostate cancer. In some embodiments, the prostate cancer is an advanced stage or an end-stage cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is a neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE). In some embodiments, the prostate cancer is CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0086] In some embodiments, the SREBP1 inhibitor is administered in combination with another SREBP1 inhibitor or one or more SREBP1 activity modulator.

[0087] In some embodiments, the SREBP1 inhibitor comprises a composition comprising a small interfering RNA specific for a messenger RNA sequence encoding the SREBP1 protein. In some embodiments, the inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a SREBP1 gene (NCBI Gene ID: 6720) or controlling transcription of a SREBP1 gene.

[0088] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment.

[0089] In some embodiments, the anti-androgen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide.

[0090] In some embodiments, the anti-androgen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

[0091] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with radiation therapy.

[0092] In some embodiments, the administration of the SREBP1 inhibitor to the subject in combination with an anti-androgen treatment decreases tumor size. In some embodiments, the administration of the SREBP1 inhibitor to the subject in combination with an antiandrogen treatment decreases tumor number. In some embodiments, the administration of the SREBP1 inhibitor to the subject in combination with an anti-androgen treatment prevents cancer metastasis.

[0093] In another aspect, provided herein is a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a SCD1 inhibitor.17ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025

[0094] In some embodiments, the prostate cancer is Stage 0 prostate cancer. In some embodiments, the prostate cancer is Stage I prostate cancer. In some embodiments, the prostate cancer is Stage II prostate cancer. In some embodiments, the prostate cancer is Stage III prostate cancer. In some embodiments, the prostate cancer is Stage IV prostate cancer. In some embodiments, the prostate cancer is early-stage prostate cancer. In some embodiments, the prostate cancer is an advanced stage or an end-stage cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is a neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE). In some embodiments, the prostate cancer is CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0095] In some embodiments, the SCD1 inhibitor is administered in combination with another SCD1 inhibitor or one or more SCD1 activity modulator.

[0096] In some embodiments, the SCD1 inhibitor comprises a composition comprising a small interfering RNA specific for a messenger RNA sequence encoding the SCD1 protein. In some embodiments, the inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a SCD1 gene (NCBI Gene ID: 6319) or controlling transcription of a SCD1 gene.

[0097] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment.

[0098] In some embodiments, the anti-androgen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide.

[0099] In some embodiments, the anti-androgen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

[0100] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with radiation therapy.

[0101] In some embodiments, the administration of the SCD1 inhibitor to the subject in combination with an anti-androgen treatment decreases tumor size. In some embodiments, the administration of the SCD1 inhibitor to the subject in combination with an anti-androgen treatment decreases tumor number. In some embodiments, the administration of the SCD1 inhibitor to the subject in combination with an anti-androgen treatment prevents cancer metastasis.18ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025

[0102] In another aspect, provided herein is a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a FASN inhibitor.

[0103] In some embodiments, the prostate cancer is Stage 0 prostate cancer. In some embodiments, the prostate cancer is Stage I prostate cancer. In some embodiments, the prostate cancer is Stage II prostate cancer. In some embodiments, the prostate cancer is Stage III prostate cancer. In some embodiments, the prostate cancer is Stage IV prostate cancer. In some embodiments, the prostate cancer is early-stage prostate cancer. In some embodiments, the prostate cancer is an advanced stage or an end-stage cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is a neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE). In some embodiments, the prostate cancer is CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0104] In some embodiments, the FASN inhibitor is administered in combination with another FASN inhibitor or one or more FASN activity modulator.

[0105] In some embodiments, the FASN inhibitor comprises a composition comprising a small interfering RNA specific for a messenger RNA sequence encoding the FASN protein. In some embodiments, the inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a. FASN gene (NCBI Gene ID: 2194) or controlling transcription of a FASN gene.

[0106] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment.

[0107] In some embodiments, the anti-androgen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide.

[0108] In some embodiments, the anti-androgen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

[0109] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with radiation therapy.

[0110] In some embodiments, the administration of the FASN inhibitor to the subject in combination with an anti-androgen treatment decreases tumor size. In some embodiments, the administration of the FASN inhibitor to the subject in combination with an anti-androgen19ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 treatment decreases tumor number. In some embodiments, the administration of the FASN inhibitor to the subject in combination with an anti-androgen treatment prevents cancer metastasis.[OHl] In another aspect, provided herein is a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a ACSL3 inhibitor.

[0112] In some embodiments, the prostate cancer is Stage 0 prostate cancer. In some embodiments, the prostate cancer is Stage I prostate cancer. In some embodiments, the prostate cancer is Stage II prostate cancer. In some embodiments, the prostate cancer is Stage III prostate cancer. In some embodiments, the prostate cancer is Stage IV prostate cancer. In some embodiments, the prostate cancer is early-stage prostate cancer. In some embodiments, the prostate cancer is an advanced stage or an end-stage cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is a neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE). In some embodiments, the prostate cancer is CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0113] In some embodiments, the ACSL3 inhibitor is administered in combination with another ACSL3 inhibitor or one or more ACSL3 activity modulator.

[0114] In some embodiments, the ACSL3 inhibitor comprises a composition comprising a small interfering RNA specific for a messenger RNA sequence encoding the ACSL3 protein. In some embodiments, the inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a ACSL3 gene (NCBI Gene ID: 2181) or controlling transcription of aACSL3 gene.

[0115] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment.

[0116] In some embodiments, the anti-androgen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide.

[0117] In some embodiments, the anti-androgen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

[0118] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with radiation therapy.20ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025

[0119] In some embodiments, the administration of the ACSL3 inhibitor to the subject in combination with an anti-androgen treatment decreases tumor size. In some embodiments, the administration of the ACSL3 inhibitor to the subject in combination with an antiandrogen treatment decreases tumor number. In some embodiments, the administration of the ACSL3 inhibitor to the subject in combination with an anti-androgen treatment prevents cancer metastasis.

[0120] In another aspect, provided herein is a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a MBOAT2 inhibitor.

[0121] In some embodiments, the prostate cancer is Stage 0 prostate cancer. In some embodiments, the prostate cancer is Stage I prostate cancer. In some embodiments, the prostate cancer is Stage II prostate cancer. In some embodiments, the prostate cancer is Stage III prostate cancer. In some embodiments, the prostate cancer is Stage IV prostate cancer. In some embodiments, the prostate cancer is early-stage prostate cancer. In some embodiments, the prostate cancer is an advanced stage or an end-stage cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is a neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE). In some embodiments, the prostate cancer is CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0122] In some embodiments, the MBOAT2 inhibitor is administered in combination with another MBOAT2 inhibitor or one or more MBOAT2 activity modulator.

[0123] In some embodiments, the MBOAT2 inhibitor comprises a composition comprising a small interfering RNA specific for a messenger RNA sequence encoding the MBOAT2 protein. In some embodiments, the inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a MBOAT2 gene (NCBI Gene ID: 129642) or controlling transcription of &MBOAT2 gene.

[0124] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment.

[0125] In some embodiments, the anti-androgen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide.21ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025

[0126] In some embodiments, the anti-androgen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

[0127] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with radiation therapy.

[0128] In some embodiments, the administration of the MBOAT2 inhibitor to the subject in combination with an anti-androgen treatment decreases tumor size. In some embodiments, the administration of the MBOAT2 inhibitor to the subject in combination with an antiandrogen treatment decreases tumor number. In some embodiments, the administration of the MBOAT2 inhibitor to the subject in combination with an anti-androgen treatment prevents cancer metastasis.

[0129] In another aspect, provided herein is a method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a GPX4 inhibitor.

[0130] In some embodiments, the prostate cancer is Stage 0 prostate cancer. In some embodiments, the prostate cancer is Stage I prostate cancer. In some embodiments, the prostate cancer is Stage II prostate cancer. In some embodiments, the prostate cancer is Stage III prostate cancer. In some embodiments, the prostate cancer is Stage IV prostate cancer. In some embodiments, the prostate cancer is early-stage prostate cancer. In some embodiments, the prostate cancer is an advanced stage or an end-stage cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is a neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE). In some embodiments, the prostate cancer is CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0131] In some embodiments, the GPX4 inhibitor is administered in combination with another GPX4 inhibitor or one or more GPX4 activity modulator.

[0132] In some embodiments, the GPX4 inhibitor comprises a composition comprising a small interfering RNA specific for a messenger RNA sequence encoding the GPX4 protein. In some embodiments, the inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a GPX4 gene (NCBI Gene ID: 2879) or controlling transcription of a GPX4 gene.22ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025

[0133] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment.

[0134] In some embodiments, the anti-androgen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide.

[0135] In some embodiments, the anti-androgen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

[0136] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with radiation therapy.

[0137] In some embodiments, the administration of the GPX4 inhibitor to the subject in combination with an anti-androgen treatment decreases tumor size. In some embodiments, the administration of the GPX4 inhibitor to the subject in combination with an anti-androgen treatment decreases tumor number. In some embodiments, the administration of the GPX4 inhibitor to the subject in combination with an anti-androgen treatment prevents cancer metastasis.

[0138] In another aspect, the invention relates to a method of treating or preventing prostate cancer in a subject in need thereof, comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a first inhibitor and a therapeutically effective amount of a second inhibitor, wherein the first inhibitor modulates the expression of a component of an intrinsic androgen signaling pathway and the second inhibitor modulates the expression of a component of an extrinsic androgen signaling pathway.

[0139] In some the prostate cancer is an early-stage prostate cancer, a Stage I prostate cancer, a Stage II prostate cancer, a Stage III prostate cancer, a Stage IV prostate cancer, an advanced stage, or an end-stage cancer.

[0140] In some embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is a neuroendocrine prostate cancer (NEPC). In some embodiments, the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE). In some embodiments, the prostate cancer is CPRC with androgen receptor expression (CRPC-AR). In some embodiments, the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

[0141] In some embodiments, the extrinsic androgen signaling pathway occurs at least partially in a stromal microenvironment. In some embodiments, the extrinsic androgen signaling pathway occurs partially in a stromal microenvironment and partially within a prostate luminal epithelial cell. In some embodiments, the intrinsic androgen signaling23ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 pathway occurs at least partially within a prostate luminal epithelial cell. In some embodiments, the intrinsic androgen signaling pathway occurs within a prostate luminal epithelial cell.

[0142] In some embodiments, the first inhibitor comprises a NKX3.1 inhibitor, a FASN inhibitor, a ACSL3 inhibitor, a MBOAT2 inhibitor, GPX4 inhibitor, or any combination thereof.

[0143] In some embodiments, the FASN inhibitor comprises a TVB-2640 (Pub Chem ID: 66548316) compound, which can also be known as Denifanstat. TVB-2640 can be referred to as its systemic name: 4-[l-[4-cyclobutyl-2-methyl-5-(5-methyl-lH-l,2,4-triazol-3- yl)benzoyl]piperidin-4-yl]benzonitrile. In some embodiments, TVB-2640 comprises, consists essentially of, or consists of the structure:, or analogs or pharmaceutically acceptable salts thereof.

[0144] In some embodiments, the FASN inhibitor comprises a TVB-3664 (Pub Chem ID: 129101638) compound. TVB-3664 can be referred to as its systemic name: 4-[l-[5-[5- (methoxymethyl)-2-(trifluoromethyl)-lH-imidazol-4-yl]-2,4-dimethylbenzoyl]azeti din-3- yl]benzonitrile. In some embodiments, TVB-3664 comprises, consists essentially of, or consists of the structure:, or analogs or pharmaceutically acceptable salts thereof.

[0145] In some embodiments, the GPX4 inhibitor comprises a RSL3 (Pub Chem ID: 89084603) compound. RSL3 can be referred to as its systemic name: methyl (lS,3R)-2-(2-24ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 chi oroacetyl)-l-(4-m ethoxy carbonylphenyl)- 1,3, 4,4a, 9, 9a-hexahydropyrido[3,4-b]indole-3- carboxylate. In some embodiments, RSL3 comprises, consists essentially of, or consists ofthe structure:, or analogs or pharmaceutically acceptable salts thereof.

[0146] In some embodiments, the GPX4 inhibitor comprises a ML162 (Pub Chem ID: 3689413) compound. ML162 can be referred to as its systemic name: 2-(3-chloro-N-(2- chloroacetyl)-4-methoxyanilino)-N-(2-phenylethyl)-2-thiophen-2-ylacetamide. In some embodiments, ML162 comprises, consists essentially of, or consists of the structure:, or analogs or pharmaceutically acceptable salts thereof.

[0147] In some embodiments, the first inhibitor comprises a composition comprising an inhibitory RNA for a messenger RNA sequence encoding a NKX3.1 protein, an inhibitory RNA for a messenger RNA sequence encoding a FASN protein, an inhibitory RNA for a messenger RNA sequence encoding a ACSL3 protein, an inhibitory RNA for a messenger RNA sequence encoding a MBOAT2 protein, an inhibitory RNA for a messenger RNA sequence encoding a GPX4 protein, or any combination thereof.25ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025

[0148] In some embodiments, the first inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a NKX3.1 gene or controlling transcription of a NKX3.1 gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a FASN gene or controlling transcription of a FASN gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding aACSL3 gene or controlling transcription of aACSL3 gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding aMBOAT2 gene or controlling transcription of aMBOAT2 gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a GPX4 gene or controlling transcription of a GPX4 gene, or any combination thereof.

[0149] In some embodiments, the second inhibitor comprises a PTN inhibitor, a mTORCl inhibitor, a MTOR inhibitor, a SREBP1 inhibitor, a SCD1 inhibitor, or any combination thereof.

[0150] In some embodiments, the mTORCl inhibitor comprises a MTOR inhibitor which comprises Sirolimus, Everolimus, Temsirolimus, Torkinib, BEZ-235, Omipalisib, and OSI- 027. In some embodiments, the mTORCl inhibitor comprises derivatives of sirolimus, or rapalogs.

[0151] In some embodiments, the mTORCl inhibitor comprises a mTOR inhibitor which comprises Sirolimus (Pub Chem ID: 5284616). Sirolimus can be referred to as its systemic name: (lR,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-l,18-dihydroxy- 12-[(2R)-l-[(lS,3R,4R)-4-hydroxy-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy- 15,17,21,23,29,35-hexamethyl-ll,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta- 16,24,26,28-tetraene-2,3,10,14,20-pentone. In some embodiments, Sirolimus comprises, consists essentially of, or consists of the structure:, or analogs or pharmaceutically acceptable salts thereof.26ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025

[0152] In some embodiments, the mTORCl inhibitor comprises a mTOR inhibitor which comprises Everolimus (Pub Chem ID: 6442177). Everolimus can be referred to as its systemic name: (lR,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-l,18- dihydroxy-12-[(2R)-l-[(lS,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]- 19,30-dimethoxy- 15,17,21 ,23 ,29,35-hexam ethyl- 11 ,36-dioxa-4- azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone. In some embodiments, Everolimus comprises, consists essentially of, or consists of the structure:5, or analogs or pharmaceutically acceptable salts thereof.

[0153] In some embodiments, the mTORCl inhibitor comprises a mTOR inhibitor which comprises Temsirolimus (Pub Chem ID: 6918289). Temsirolimus can be referred to as its systemic name: [(lR,2R,4S)-4-[(2R)-2- [(lR,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-l,18-dihydroxy-19,30- dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-ll,36-dioxa-4- azatri cyclo[30.3.1.04, 9]hexatriaconta- 16, 24,26, 28-tetraen-12-yl]propyl]-2- methoxy cyclohexyl] 3 -hydroxy -2-(hydroxymethyl)-2-methylpropanoate. In some embodiments, Temsirolimus comprises, consists essentially of, or consists of the structure:27ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025, or analogs or pharmaceutically acceptable salts thereof.

[0154] In some embodiments, the second inhibitor comprises a composition comprising an inhibitory RNA for a messenger RNA sequence encoding a PTN protein, an inhibitory RNA for a messenger RNA sequence encoding a MTOR protein, an inhibitory RNA for a messenger RNA sequence encoding a mTORCl protein, an inhibitory RNA for a messenger RNA sequence encoding a RPTOR protein, an inhibitory RNA for a messenger RNA sequence encoding a SREBP1 protein, an inhibitory RNA for a messenger RNA sequence encoding a SCD1 protein, or any combination thereof.

[0155] In some embodiments, the second inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a PTN gene or controlling transcription of a PTN gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a mTORCl gene or controlling transcription of a mTORCl gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a. MTOR gene or controlling transcription of a MTOR gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a RPTOR gene or controlling transcription of a RPTOR gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a SREBP1 gene or controlling transcription of a SREBP1 gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a SCD1 gene or controlling transcription of a SCD1 gene, or any combination thereof.

[0156] In some embodiments, the first inhibitor comprises a NKX3.1 inhibitor, a FASN inhibitor, a ACSL3 inhibitor, a MBOAT2 inhibitor, GPX4 inhibitor, or any combination28ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 thereof, and the second inhibitor comprises a PTN inhibitor, a MTOR inhibitor, a mTORCl inhibitor, a SREBP1 inhibitor, a SCD1 inhibitor, or any combination thereof.

[0157] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment. In some embodiments, the antiandrogen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide. In some embodiments, the antiandrogen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

[0158] In some embodiments, the pharmaceutical composition is administered before, after, or in combination with radiation therapy.

[0159] In some embodiments, the administration of the pharmaceutical composition to the subject in combination with an anti-androgen treatment decreases tumor size. In some embodiments, the administration of the pharmaceutical composition to the subject in combination with an anti-androgen treatment decreases tumor number. In some embodiments, the administration of the pharmaceutical composition to the subject in combination with an anti-androgen treatment prevents cancer metastasis.

[0160] In some embodiments, the subject is human.

[0161] In some embodiments, NKX3.1, FASN, ACSL3, MBOAT2, GPX4, PTN, MTOR, mTORCl, RPTOR, SREBP1, SCD1, or any combination thereof is inhibited through genetic alterations. In certain aspects, described herein are nucleic acids encoding RNAs of interest, which includes, but is not limited to an interfering RNA (iRNA), and variants thereof, that can silence a target gene, such as NKX3.1, FASN, ACSL3, MBOAT2, GPX4, PTN, MTOR, mTORCl, RPTOR, SREBP1, SCD1, or any combination thereof. An iRNA can down- regulate the expression of a target gene, e.g., NKX3.1, FASN, ACSL3, MBOAT2, GPX4, PTN, MTOR, mTORCl, RPTOR, SREBP1, SCD1, or any combination thereof. An inhibitory RNA (iRNA) may act by one or more of a number of mechanisms, including post- transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre- transcriptional or pre-translational mechanisms. An iRNA can be a double stranded (ds) iRNA. A ds iRNA includes more than one, and in certain embodiments two, strands in which interchain hybridization can form a region of duplex structure. A strand refers to a contiguous sequence of nucleotides (including non-naturally occurring or modified nucleotides). At least one strand can include a region which is sufficiently complementary to a target RNA. Such strand is termed the antisense strand. A second strand comprised in the dsRNA which comprises a region complementary to the antisense strand is termed the sense29ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 strand. However, a ds iRNA can also be formed from a single RNA molecule which is, at least partly; self-complementary, forming, e.g., a hairpin or panhandle structure, including a duplex region. In such case, the term strand refers to one of the regions of the RNA molecule that is complementary to another region of the same RNA molecule. Nonlimiting examples of inhibitory RNA include miRNA, siRNA, shRNA, and piRNA. iRNA as described herein, including ds iRNA and siRNA, can mediate silencing of a gene, e.g., by RNA degradation. In certain embodiments, the gene to be silenced is NSD2. In some embodiments, NSD2 translation is downregulated or inhibited via small interfering RNA targeting the NSD2 mRNA.

[0162] In certain embodiments, the CRIPSR cassette comprises an oligonucleotide comprising a guide RNA (gRNA) or single guide RNA (sgRNA). The CRISPR / Cas9 gene editing technique promotes a new human gene therapy strategy by editing DNA at pre-chosen sites without altering the endogenous regulation of target genes. This system consists of two key components: Cas9 protein and a guide RNA, e.g., a single guide RNA (sgRNA), as well as a correction template when needed. In some embodiments, a CRISPR cassette refers to a DNA construct that contains all necessary elements for CRISPR-based gene editing, typically including the sgRNA sequence, the Cas9 coding region, and regulatory elements for expression. sgRNA contains two components: a 17-20 nucleotide sequence termed CRISPR RNA that is complementary to the target DNA region, and a tracrRNA (trans-activating CRISPR RNA) that serves as the binding scaffold for a Cas nuclease. In some embodiments, the CRISPR cassette comprises the sgRNA sequence, wherein the other elements for CRISPR-based gene editing are provided separately. The cassette enables delivery and expression of the CRISPR components in target cells, facilitating precise genome editing. The sgRNA recognizes the target DNA and guides the Cas9 nuclease to the region for editing.

[0163] The inhibitors or compositions used to treat or prevent prostate cancer described herein may be formulated into pharmaceutical compositions or formulations using one or more pharmaceutically acceptable excipients, carriers, or diluents. These pharmaceutical compositions or formulations may be designed for various routes of administration, including but not limited to oral, intravenous, intramuscular, subcutaneous, intranasal, intraperitoneal, intradermal, or topical delivery. Suitable excipients may include stabilizers, preservatives, solubilizers, emulsifiers, buffers, and agents that enhance bioavailability or control release kinetics. Such pharmaceutical compositions or formulations may be tailored to improve stability, shelf-life, patient compliance, and therapeutic performance. The pharmaceutical compositions or formulations may be provided in dosage forms such as tablets, pills,30ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 capsules, solutions, suspensions, emulsions, injectables, or transdermal patches, depending on the intended use and delivery method.

[0164] The dosage and administration of the compositions or formulations described herein may vary depending on several factors, including but not limited to the nature and severity of the condition being treated, the route of administration, the formulation used, and the individual characteristics of the subject such as age, weight, sex, and overall health status. Determining the appropriate dosing regimens may be established based on clinical guidelines, preclinical data, and routine experimentation. Dosages may be adjusted to achieve optimal therapeutic efficacy while minimizing potential side effects. In certain embodiments, the compositions or formulations may be administered in a single dose, multiple doses, or as part of a continuous or intermittent dosing schedule.Dual pathway as the target for prostate cancer prevention and treatment.

[0165] Described herein is the discovery that the primary mode of cell death following castration is not apoptosis, but is instead ferroptosis, an iron-dependent form of regulated cell death due to lipid peroxidation. Described herein is the integration of two independent pathways that are activated by androgen receptor (AR) to suppress fenoptosis and govern castration-response. One of these pathways is intrinsic to prostate luminal epithelial cells, whereas the other pathway involves extrinsic signaling from the stromal microenvironment. This dual regulatory pathway provides a fail-safe mechanism to safeguard the prostate from undergoing accidental ferroptosis due to tissue insults such as inflammation.

[0166] Conversely, however, this dual pathway mechanism also suggests that castrationresistance, once acquired, is particularly difficult to overcome, since it may be necessary to block both arms of the pathway of castration-response. This concept of dual inhibition can explain why inhibitors of the AKT-mTOR pathway, which, as described herein, is a central part of the extrinsic signaling pathway, have displayed limited efficacy in clinical trials, despite the key role of this pathway in mediating castration-resistance. Instead, combined inhibition of intrinsic and extrinsic pathways represents an alternative approach for targeting castration resistance.The central role of androgen signaling in the prostate.

[0167] Since the seminal work of Huggins and Hodges in the 1940s (see Ref 1), it has been widely recognized that androgen signaling plays a fundamental role in nearly all aspects of prostate development, homeostasis, and cancer (see Refs 2, 3, and 4). In particular,31ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 androgen-deprivation therapy (ADT) through chemical or surgical castration leads to regression of normal prostate tissue as well as treatment-naive primary prostate tumors. Even when prostate tumors recur following androgen-deprivation therapy in the form of castrationresistant prostate cancer (CRPC), these tumors usually remain dependent on the activity of androgen receptor (AR), which has led to the development and clinical application of potent second-generation AR antagonists (see Ref 5). Thus, inhibition of AR activity has represented the mainstay of prostate cancer treatment for the past 80 years.

[0168] Despite the central role of androgen-deprivation in prostate cancer therapy, the molecular basis of castration-sensitivity in the prostate has remained poorly understood.Early work elucidated the time course of prostate tissue regression following castration in the mouse and rat, and demonstrated profound decreases in tissue size as well as epithelial cell size during regression (see Ref 6). Several of these studies showed that cell death in luminal epithelial cells due to apoptosis could be detected during regression (see Refs 7-10). Similar studies have suggested that apoptosis occurs as an early response to androgen-deprivation in the human prostate (see Ref 11). However, the calculated apoptotic index in the regressing prostate described in these early studies appears to be substantially lower than could account for the extensive cell death observed during regression. Despite this key knowledge gap, the mechanism(s) of cell death during prostate regression have not been further investigated in detail, even though in intervening years many additional forms of programmed cell death have been described.Intrinsic and extrinsic responses to androgen-deprivation.

[0169] In the normal prostate, AR is widely expressed in epithelial, stromal, as well as immune cells. Classically, histological and ultrastructural analyses have described three major epithelial cell types in the prostate: luminal cells, basal cells, and rare neuroendocrine cells (see Refs 3 and 12); furthermore, recent single-cell RNA-sequencing (scRNA-seq) analyses have demonstrated extensive heterogeneity within the luminal epithelial population. Luminal epithelial cells represent a major target of castration-induced changes in the prostate epithelium, but it has been unknown whether the requirement for AR activity to maintain tissue integrity is extrinsic and / or intrinsic to the epithelium. In principle, AR may function in the stroma, epithelium, or both, to promote survival of luminal epithelial cells, perhaps through production of “anti -death” signals (FIG. 1A). Following androgen-deprivation, the loss of such signals leads to luminal cell death and tissue regression (FIG. IB), whereas in32ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 the castration-resistant state, alternative pathways activated during tumorigenesis may bypass the requirement for AR to promote survival (FIG. 1C).

[0170] Notably, pioneering studies by the Cunha laboratory elicited the key role of stromal-epithelial interactions in prostate development and cancer, using tissue recombination approaches in mice (Refs 13 and 14). Subsequent studies in vivo have shown that Cre- mediated deletion of AR in epithelial cells or in stromal fibroblasts leads to impaired prostate outgrowth, indicating a requirement of AR in both compartments for normal development (Refs 15 and 16). More recent work has shown that stromal AR activity plays an essential role for prostate epithelial outgrowth during development (Refs 17 and 18). Similarly, studies from the Cunha lab using tissue recombinants suggested a key role for stromal signals in inducing apoptosis following castration (Ref 19). However, due to technical limitations preventing the analysis of AR-null stroma in tissue recombinants, this study could not rule out a role for intrinsic AR-dependent signaling within the epithelium in promoting cell death following androgen-deprivation. Moreover, the overall apoptotic index measured in this study was significantly lower (approximately 2%) than could account for the extensive cell death during regression.Ferroptosis as a cell death mechanism.

[0171] Ferroptosis was first described by Brent Stockwell’s group as an iron-dependent form of regulated cell death due to lipid peroxidation (see Ref 20). An overview of the ferroptosis pathway is provided in FIG. 2A (see also Ref 97) and the markers of ferroptosis is presented in FIG. 2B (see also Ref 22). Subsequent studies have distinguished ferroptosis from other forms of cell death, have described detailed mechanisms that regulate ferroptosis, and have elucidated its roles in a wide range of disease and cancer contexts (Refs 21-23). In particular, ferroptosis is driven by accumulation of membrane phospholipids containing polyunsaturated fatty acids (PUFAs), which can readily undergo peroxidation by reactive oxygen species. Disruption of the balance between monounsaturated fatty acids (MUFAs) and PUFAs thus plays a critical role in determining susceptibility to ferroptosis. Maintenance of redox homeostasis also plays a central role, particularly through the function of glutathione (GSH) in reduction of reactive oxygen species (ROS) as well as its role as a cofactor for the glutathione peroxidase GPX4, which inhibits ferroptosis by reduction of phospholipid hydroperoxides.

[0172] To date, however, there have been few examples of a role for ferroptosis in normal physiological contexts. In the Examples below, it is shown that ferroptosis represents the33ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 primary mechanism for cell death due to androgen-deprivation in the normal prostate. Notably, the response to castration requires the loss of AR activity in the prostate stroma through an extrinsic pathway, as well as the loss of AR activity within luminal epithelial cells through an intrinsic pathway (FIG. 2C). The inactivation of both the extrinsic and intrinsic AR signaling pathways is required to trigger extensive tissue regression and ferroptosis following androgen-deprivation. The integration of dual pathways provides a fail-safe mechanism to prevent prostate regression in contexts of tissue damage that do not involve decreased AR signaling, such as formation of ROS during inflammation.

[0173] Referring to FIG. 2C, a more detailed illustration of the dual pathway mechanism for induction of ferroptosis following androgen-deprivation is provided. An extrinsic AR signaling pathway starting from stroma (shown in dark gray) integrates with an intrinsic AR pathway within luminal cells (in light gray) to suppress ferroptosis in the hormonally-intact prostate. Intrinsic AR signaling controls the expression of NKX3.1, which together with AR regulates downstream transcriptional targets, including GPX4 and regulators of MUFA synthesis. Extrinsic signaling mediated by stromal PTN also regulates MUFA synthesis in luminal cells through an mTORCl-SREBPl axis. Loss of both extrinsic and intrinsic AR pathways is necessary to trigger a loss of MUFA synthesis with GPX4 downregulation to trigger lipid peroxidation and ferroptosis.***

[0174] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0175] All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art.EXAMPLES

[0176] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments34ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.Example 1 — Differing responses to castration due to luminal heterogeneity

[0177] To understand the response of luminal epithelial cells to castration, it is essential to understand the composition of luminal cell populations in the normal prostate. Recent scRNA-seq analyses have revealed considerable heterogeneity of luminal epithelial cells in the mouse prostate epithelium (see Refs 24-28), reviewed in Ref 12 (FIGS. 3A-C). FIGS. 3A-C shows the epithelial heterogeneity and proximal-distal asymmetry in the mouse prostate. FIG. 3A is an aggregated composite plot of published and new scRNA-seq datasets for mouse prostate (see Ref 31). FIG. 3B is the schematic model of prostate lobes indicating the distribution of luminal epithelial populations (see Ref 24). FIG. 3C is a dot plot of gene expression levels in each epithelial population for selected markers (see Ref 24). FIG. 3D is a sharp boundary of gene expression divides proximal from distal regions.

[0178] Similar heterogeneity has been observed in studies of benign human prostate (Refs 24-26, 29), as well as in the context of pan-tissue resources (Ref 30). In the mouse prostate, these populations are separated along the proximal-distal axis of each prostate lobe, with distal luminal cells (LumDist) representing the secretory luminal cells, and a much smaller but distinct population of luminal cells located more proximally (LumP), and a population of periurethral (PrU) cells with shared luminal and basal features adjacent to the urethra (FIGS. 3B-D). In addition, our meta-analysis of published mouse prostate scRNA-seq datasets (Ref. 31) has demonstrated clear transcriptomic differences between distal luminal cells, which correspond to traditional secretory cells, versus those that are proximal, which have progenitor properties (Refs 24 and 25). Importantly, a key regulator of prostate cell identity, the homeodomain transcription factor NKX3.1 (Ref. 32-34), is specifically expressed in distal but not proximal luminal epithelial cells (FIG. 3D). Furthermore, distal and proximal luminal populations differ in their response to castration, as distal but not proximal luminal cells primarily undergo cell death following androgen-deprivation (Refs 26, 31, and 35).Example 2 — Defining the features of castration-response in the prostate epithelium

[0179] Although numerous changes occur in the prostate during tissue regression, we will focus below on the alterations that lead to cell death of distal luminal epithelial cells after castration, as these mechanisms are most likely to have translational significance for prostate35ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 cancer. Furthermore, to ensure their biological relevance, our studies have utilized in vivo analyses to study castration-response in genetically-engineered mouse models, followed by validation in human tissues.

[0180] In our studies, we performed a time course analysis of prostate tissue regression following surgical castration in wild-type C57BL / 6 mice (FIG. 4G). We analyzed all four prostate lobes, which displayed similar results. FIGS. 4A-F show data on the anterior prostate (AP) that represent defining features of prostate regression following castration. FIG. 4A shows the dark-field and immunofluorescence (IF) staining of dissected anterior prostate (AP) lobes from wild type (WT) C57BL / 6 mice that were hormonally intact or castrated for the indicated number of days. FIGS. 4B-C show plots of prostate lobe weight (FIG. B) and luminal cell size (FIG. C) in AP lobes from intact and castrated WT mice. Cell size was calculated by measuring Cytokeratin 8 (CK8)-stained cell areas (pm2). FIG. 4D are plots of cell size using forward scatter area (FSC-A) of sorted distal and proximal YFP- positive luminal cells from tam oxifen-treated Nkx3. iCreERT2 / +;R26R-YFP mice (NY), which labels distal luminal cells, and Krl7!'re-RT2; R26R-YFP mice (KY), which labels proximal luminal cells. FIG. 4E shows a plot of luminal cell number in AP lobes from intact and castrated WT mice. Luminal cell numbers were determined by isolation of luminal cells by flow sorting with the protocol in Ref 24 using CountB right Counting Beads (ThermoFisher Scientific); values were normalized to the luminal cell number in intact mice. P-values were calculated by t tests. FIG. 4F are scatterplots of scRNA-seq data for AP luminal cells from hormonally intact or castrated mice, showing the distal luminal (x axis) and proximal luminal (y axis) intact signature score (z score) for each cell (dot) assigned to luminal distal (blue) or luminal proximal (red) at each time point. Dot color intensity is scaled by the strength of a XGBoost classifier assignment probability for each dot’s assigned class (color bar).

[0181] Our analyses show that we can define prostate regression through changes in lobe size, distal luminal cell size, distal luminal cell number, and luminal transcriptomic state, which notably did not occur in a coordinated manner. Thus, we could readily observe progressive decreases in lobe size and weight starting at 3 days after castration and continuing until the end of prostate regression by 28 days (FIGS. 4A, B). We also measured luminal cell size from tissue sections, which progressively decreased to a greater extent in distal luminal cells than in proximal luminal cells (FIGS. 4A, C, and D); these changes in cell size are due to autophagy (data not shown).

[0182] In contrast, we found that decreases in luminal cell number as measured by flow cytometry followed a different time course. We observed a transient increase in luminal cell36ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 number at 3 days after castration, and minimal changes in cell number at 7 days, but a profound loss of luminal cells by 28 days (FIG. 4E). The increased number of luminal cells at 3 days is due to a normal function of AR in suppressing epithelial cell proliferation, as conditional deletion of AR in luminal epithelial cells has been shown to induce transient cell proliferation (Refs 36-38). Finally, we observed a transcriptomic shift in cell state in the small percentage of distal luminal cells that survive castration, as their gene expression programs become proximalized, most closely resembling normal PrU cells (Refs 26, 31). These surviving distal luminal cells acquire expression of the proximal marker PPP1R1B (Fig. 4A), and display a shift toward proximal cell states at the single-cell transcriptomic level (FIG. 4F). A similar but smaller transcriptomic shift occurs in proximal luminal cells towards a PrU-like state (Ref 31) (FIG. 4F). Notably, distal luminal cells in the regressed state display elevated expression oi A lox 12 and pathways that suppress reactive oxygen species (Ref 35), consistent with a potential ferroptotic response.Example 3 — Luminal cell death following castration is mediated by ferroptosis

[0183] To examine cell death mechanisms following castration, we examined luminal proliferation and apoptosis in the mouse prostate after castration. Co-staining Ki67 and luminal cell specific marker CK8 show a transient cell proliferation within 2 days after castration (FIG. 11A). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining shows an induction of apoptosis in luminal cells after castration, with a peak at 3 days after castration (FIG. 11A). However, the apoptotic ratio, which is around 0.5-2% is inconsistent with the loss of luminal cells after castration, where more than 90% luminal cells undergo cell death, indicating apoptosis is not the major type of cell death that mediates castration induced prostate regression (FIGS. 11B-C, 4E).

[0184] To identify the key pathway for distal luminal cell death, we used an in vivo genetic approach. FIG. 5A provides the luminal cell numbers in AP lobes at day 28 after castration. Inducible deletion of Acsl4, Casp8 and Gsdmd in luminal cells was used to block the activation of ferroptosis, apoptosis and pyroptosis, respectively, and deletion efficiency was confirmed by immunoblotting. Using the Tmprss2-CreERT2driver for inducible Cre- mediated deletion in the prostate epithelium (see Ref 39), we found that partial conditional deletion of Acsl4, a key regulator of ferroptosis (see Ref 40), could significantly rescue the cell death of distal luminal cells after castration (FIG. 5A). In contrast, inducible deletion of Casp8, which is essential for apoptosis (see Ref 41), or Gsdmd, which is essential for pyroptosis (see Refs 42 and 43), did not result in significant rescue of cell death (FIG. 5A).37ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025This is consistent with enrichment of a ferroptosis signature in castrated luminal cells versus intact luminal cells using a scRNA-seq dataset as shown in the ridgeline plots in FIG. 11D, which are for the distribution of gene set enrichment (GSEA) results for four cell death signatures in luminal cells, comparing scRNA-seq data for WT Cas d7 versus WT intact prostate. The shadings represent the adjusted p-values calculated by the clusterProfiler package. The experimental strategy for testing the in vivo roles of ferroptosis, apoptosis, and pyroptosis in luminal cell death after castration is shown in FIG. 5G. We also observed that treatment of wild-type mice with a potent inhibitor of lipid peroxidation, liproxstatin-1, could dramatically suppress cell death following castration (FIG. 5H).

[0185] To confirm that ferroptosis occurs during prostate tissue regression, we next performed lipidomic measurements by mass spectrometry. FIGS. 5B shows the lipidomic analysis of the most abundant poly-unsaturated fatty acids (PUFAs) in luminal cells from intact and castrated WT mice; all comparisons shown are significant (p < 0.001). These analyses demonstrated significant decreases in abundance of a wide range of polyunsaturated fatty acids (PUFAs) between control prostates and prostates at 7 days after castration (FIG. 5B), consistent with lipid peroxidation prior to the occurrence of widespread distal luminal cell death (FIG. 4E).

[0186] We also performed Western blotting to examine expression of proteins that play key roles in mono-unsaturated fatty acid (MUFA) synthesis, including regulatory components of the PI3K-mTOR-SREBPl pathway. FIG. 5C is the immunoblotting of proteins involved in mTORCl activity and MUFA synthesis in AP luminal cells from WT and castrated mice, using samples from three biological replicates. We found that many of these proteins decreased during early stages after castration, consistent with an imbalance in the MUFA / PUFA ratio that promotes ferroptosis (FIG. 5C). In contrast, we observed a strong increase in the levels of transferrin receptor (TFR1), a marker of ferroptosis (see Ref 44) (FIG. 5C)

[0187] Next, we examined changes in mitochondrial morphology by electron microscopy, which revealed substantial decreases in mitochondrial size and cristae number at 7 days after castration (FIGS. 5D-F), which are consistent with the occurrence of ferroptosis. FIG. 5D are scanning electron micrographs of distal luminal cells. The blue and red arrows indicate mitochondria from distal luminal cells of intact and castrated WT mice, respectively. FIGS. 5E-F are plots of mitochondrial cristae number (E) and area (F).

[0188] We also observed that treatment of wild-type mice with a potent inhibitor of lipid peroxidation, liproxstatin-1, could dramatically suppress cell death following castration (FIG.38ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 20255J)Taken in combination, these genetic, lipidomic, ultrastructural, and biochemical lines of evidence indicate that luminal cell death following castration is due to ferroptosis. That is, ferroptosis is a major type of cell death in luminal cells that mediates castration induced prostate regression.Example 4 — Loss of androgen receptor is needed in both the stroma and epithelium to promote cell death

[0189] To determine whether prostate regression following androgen-deprivation is due to the loss of AR function in the epithelium or stroma, or both, we employed a genetic approach for inducible deletion of Ar in vivo. To delete AR in the prostate epithelium (which we term Lum AR KO), we used the inducible Tmprss2-CreERT2driver in Tmprss2-CreERT2; ^rflox / ymiceporefficient deletion of Ar in the stroma, we used the combination of two stromal Cre drivers in Vimentin-CreERT2; Colla2-CreERT2; Ar^ox / Ymice (Str AR KO), or all three Cre drivers to delete Ar in both the epithelium and stroma (Lum+Str AR KO). FIG. 12A provides the experimental strategy and mouse genotypes for inducible deletion of AR in prostate luminal cells and / or stromal cells. FIG. 12B is the immunostaining for AR, CK5, CK8, and Vimentin (VIM) in the AP lobes of the indicated genotypes to delete AR in luminal cells, stromal cells, or both at 28 days after tamoxifen-induction. Scale bars, 50 pm.

[0190] FIG. 6 shows that androgen receptor has distinct roles in stroma and epithelium. FIGS. 6A, F are dark-field and IF images of dissected AP lobes from mice with induced deletion of Ar in luminal cells (Lum AR KO), stromal cells (Str AR KO), or both (Lum + Str AR KO), at day 28 after tamoxifen induction. FIG. 6B-D, G are plots of AP lobe weight (FIG. 6B), distal luminal cell size (FIG. 6C), proximal luminal cell size (FIG. 6G), and luminal cell number (FIG. 6D) from mice with deletion of Hr in luminal cells, stromal cells, or both at day 28 after tamoxifen induction; p-values were calculated by t tests. FIG. 6E show transcriptomic changes in AP luminal cells following Ar deletion in luminal cells, stromal cells, or both at day 28 after tamoxifen induction. Scatterplots were generated from scRNA-seq data as in FIG. 4F; the WT intact and WT Cas d28 plots are identical to those in FIG. 4F

[0191] We found that Lum AR KO could induce decreased lobe weight, cell size, and transcriptomic shift, whereas Str AR KO could only induce decreased lobe weight and cell size (FIG. 6A-C, E). However, the combination of both luminal and stromal Ar deletion (Lum+Str AR KO) was necessary to achieve decreased luminal cell death (FIG. 6D), indicating that loss of AR activity in both the epithelium and stroma is necessary to induce39ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 ferroptosis. Thus, ferroptosis is regulated by both an extrinsic as well as an intrinsic pathway in distal luminal epithelial cells. Accordingly, inhibition of both intrinsic and extrinsic androgen signal is required to induced ferroptosis in luminal cells for prostate regression.Example 5 — The intrinsic castration-response pathway is mediated by Nkx3.1

[0192] The homeodomain transcription factor NKX3.1 is a strong candidate regulator for the intrinsic pathway of castration-response. NKX3.1 is expressed in distal but not proximal luminal cells (FIG. 3B), and its expression is known to be regulated by AR (see Refs 45 and 46); furthermore, NKX3.1 acts as a cofactor for AR in binding to a subset of canonical AR targets (see Ref 47). Therefore, we investigated the phenotypes of Nkx3.1 null mutant mice (see Ref 32), which had not previously been analyzed in this context.

[0193] As shown in FIG. 7, intrinsic signaling is mediated by activities of NKX3.1. FIGS. 7A-B show AP lobe weight (FIG. 7A) and luminal cell number (FIG. 7B) from mice with homozygous null Nkx3.1 mutation and / or stromal Ar deletion at day 28 days after tamoxifen induction. FIGS. 7C-D are measurements of GSH (FIG. 7C) and GSH / GSSG ratio (FIG. 7D) in AP luminal cells from WT and Nkx3.1 mutant mice; p-values were calculated by t tests. FIG. 7E are immunoblottings of proteins involved in mono-unsaturated fatty acid (MUFA) synthesis in AP luminal cells isolated from WT and Nkx3.1 mutant mice, using samples from three biological replicates. FIGS. 7F, G show the abundance of representative MUFA-phospholipids in luminal cells from WT intact and Cas d7 mice (FIG. 7F) and from mice with induced deletion of Ar at day 7 after tamoxifen induction (FIG. 7G), with each dot corresponding to lipids extracted from combined luminal cells from 5 mice. FIG. 7H shows AR and NKX3.1 ChlP-seq signal tracks near Gpx4 and MUFA synthesis gene loci in mouse prostate datasets, indicating that the putative enhancers for Acsl3, Mboat2, Gpx4. and Fads2 are bound by NKX3.1 and AR.

[0194] We found that Nkx3.1 mutant prostates did not display alterations in lobe weight or luminal cell number as expected, but in combination with inducible stromal Ar deletion (Str AR KO), we found loss of luminal cells as well as a further decrease in lobe weight (FIGS. 7A-B, I-J) and luminal cell size (FIGS. 7K-L). Notably, Nkx3.1 null mutants display alterations of redox homeostasis with decreased GSH level and GSH / GSSG ratio (FIGS. 7C- D), consistent with previous work by the Abate-Shen lab showing increased ROS levels in Nkx3.1 mutants (see Refs 48, 49). Distal luminal cells in Nkx3.1 mutants also displayed a transcriptomic shift towards proximal states, resembling distal luminal cells from Lum Hr KO prostates (FIG. 7M). Furthermore, we observed that the levels of regulators of MUFA40ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 synthesis such as ACSL3 and MBOAT2, as well as the key ferroptosis regulator GPX4, were decreased in Nkx3.1 mutants (FIG. 7E), suggesting that these genes may be targets of NKX3.1 transcriptional activity. These results indicate that NKX3.1 suppresses ferroptosis in the hormonally intact prostate, and plays a central role in the intrinsic pathway for castrationresponse.Example 6 — Extrinsic castration-response is mediated by a PTN-mTORCl-SREBP pathway

[0195] To identify potential stromal signals downstream of AR that suppress ferroptosis in the luminal epithelium, we used scRNA-seq data to screen known stromal signaling factors for their downregulation following castration. This screen identified several candidate signaling factors (FIG. 8H), one of which was Pleiotrophin (PTN), which is a secreted heparin-binding factor that is upregulated by AR in the prostate stroma and can signal to prostate epithelial cells (see Ref 50). The other candidate identified was Insulin-like Growth Factor 1 (IGF1), but our investigation suggests that Igfl is not required for castration induced prostate regression. Luminal deletion of IGF 1 receptor (Igflr) did not affect SREBP1 or p- S6 levels (FIG. 81), and had minimal phenotypes relative to Lum Ar KO prostates, with no significant effect on lobe weight and luminal cell number (FIGS. 8J-K).

[0196] To test whether PTN might represent the extrinsic signal that suppresses ferroptosis in the hormonally intact prostate, we used inducible stromal deletion of a conditional Ptn allele in Vimentin-CreERP2; Colla2-CreERT2; Ptiox / floxmice (Str Ptn KO) together with Nkx3.1 null mutants to determine whether this combination of extrinsic and intrinsic mediators could recapitulate prostate regression in the absence of androgendeprivation or AR deletion.

[0197] FIG. 8 shows that extrinsic signaling utilizes a PTN-mTORCl-SCDl axis. FIGS. 8A-B show AP lobe weight (FIG. 8A) and luminal cell number (FIG. 8B) from mice with stromal deletion of Ptn and / or Nkx3.1 homozygous mutation at 28 days after tamoxifen induction. FIG. 8C are the immunoblottings of proteins involved in mTORCl activity and MUFA synthesis in luminal cells from WT and stromal deleted Ptn mice, using samples from three biological replicates. FIGS. 8D-E show the AP lobe weights (FIG. 8D) and luminal cell numbers (FIG. 8E) from mice with luminal deletion of Raptor and / or Ar at 28 days after tamoxifen induction; p-values were calculated by t tests. FIGS. 8F are the immunoblottings of proteins involved in mTORCl activity and MUFA synthesis in WT and Raptor deleted luminal cells, using samples from three biological replicates.41ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025

[0198] Our results show that stromal deletion of Ptn alone had a modest effect on lobe weight, but no effect on luminal cell number, whereas the combination of stromal Ptn deletion and Nkx3.1 loss resulted in a significant decrease in cell number (FIGS. 8A-B), indicating that E / / ? is a key component of the extrinsic signal. Although the downstream signaling pathway for PTN has not been well-characterized, we found that inducible stromal deletion of Ptn resulted in decreased levels of pS6 and SREBP1 in luminal epithelial cells (FIG. 8C), suggesting that PTN is an upstream regulator of the mTORCl-SREBPl pathway that governs fatty acid synthesis and suppresses ferroptosis (Ref 51).

[0199] Based on these findings, we next investigated whether the mTORCl-SREBPl pathway is a central downstream component of the extrinsic pathway that mediates castration-response. To evaluate this possibility, we performed inducible luminal-specific deletion of Raptor, a constitutive component of the mTORCl complex, using the Tmprss2- CreERT2driver. We found that luminal-specific deletion of Raptor resulted in decreased lobe weight, but had no significant effect on luminal cell number, whereas the combined luminal deletion of Raptor and Ar resulted in a strong decrease in luminal cell number (FIGS. 8D-E, G). Luminal deletion of Raptor resulted in decreased levels of pS6, SREBP1, and SCD1, but did not affect FASN (FIG. 8F). Taken together, these data indicate that stromal signaling from PTN through an mTORCl -SCD1 pathway in luminal cells represents the extrinsic pathway that suppresses ferroptosis in the hormonally-intact prostate. Our findings are particularly suggestive given the substantial body of literature demonstrating a central role for the PTEN-AKT-mTOR pathway in castration-resistance (Refs 52 and 53). Accordingly, PI3K-AKT-mTORCl-SREBPl axis integrates extrinsic androgen signal in regulating castration induced prostate regression.Example 6 — Evidence for ferroptosis in human prostate cancer following ADT

[0200] To validate our findings in mouse models to human prostate, we examined the expression of ferroptosis markers in tissue samples from primary prostate cancer patients that have undergone androgen-deprivation. FIG. 9 shows evidence for ferroptosis in human prostate after ADT or enzalutamide treatment. FIG. 9A is the immunohistochemical (H4C) staining for AMACR, a marker of prostate tumors, and TFR1, a marker of ferroptosis, in benign prostate and prostate tumors from treatment-naive patients or patients who received neoadjuvant ADT prior to transurethral resection or prostatectomy. In analyses of prostate tissue samples from two patients who had received neo-adjuvant ADT prior to surgery, as well as two age-matched treatment-naive control patients, we found upregulation of42ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 transferrin receptor (TFR1), a marker of ferroptosis (Ref 44), in both benign as well as tumor tissues after ADT (FIG. 9A).

[0201] We also examined whether there was evidence for ferroptosis following antiandrogen treatment of CRPC. For this purpose, we examined a published dataset of bulk RNA-seq data of biopsies from metastatic CRPC patients who were either responders or nonresponders to enzalutamide treatment (see Ref 54). FIG. 9B is the gene set enrichment analysis (GSEA) using bulk RNA-seq data from tumor biopsy samples from mCRPC patients who were responders or non-responders to enzalutamide treatment (see Ref 54). Gene set enrichment analyses using expression signatures for ferroptosis (see Refs 23, 55) and apoptosis (Ref 56) showed significant enrichment for the ferroptosis signature but not apoptosis signature in enzalutamide responders relative to non-responders (FIG. 9B). In combination, these preliminary data suggest that ferroptosis can occur following ADT in primary prostate tumors as well as after anti-androgen treatment of CRPC.

[0202] We have shown that luminal epithelial cells display a profound difference in their castration-response depending on their cell state. In brief, distal luminal cells are highly castration-sensitive and express the key intrinsic pathway regulator NKX3.1, whereas proximal luminal cells are more castration-resistant and lack NKX3.1 expression; however, both distal and proximal luminal cells express AR. Following castration, the vast majority of distal luminal cells undergo cell death due to ferroptosis, but surviving distal luminal cells undergo a transcriptomic shift to a more proximal state that is inherently castration-resistant.

[0203] Previous studies of the dynamic response to castration have focused on early time points (day 3, day 7) after castration (Refs 25, 26, 31), but our analyses of luminal cell numbers following castration (FIG. 4E) suggest that the time frame of ferroptosis induction extends over a much longer period during prostate regression.

[0204] Our findings suggest that several key enzymes responsible for MUFA biosynthesis (ACSL3, MB0AT2) as well as other regulators of ferroptosis (e.g., GPX4) are regulated by NKX3.1 activity in distal luminal epithelial cells (FIG. 2; FIG. 7E). Furthermore, MB0AT2 has been shown to be regulated directly by AR in prostate cancer cells (Ref 58), while NKX3.1 itself is an AR downstream target (Ref 46) and can act as a transcriptional co-factor for AR (Ref 47).

[0205] We have examined chromatin accessibility at known AR target genes, such as Nkx3.1 itself, using a multi ome dataset that we generated from wild-type adult prostate (FIG. 10). These data demonstrate that chromatin accessibility can differ between proximal versus distal luminal cells, and support the feasibility of our proposed analyses. FIG. 10 shows the43ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 multiome analysis of chromatin accessibility in prostate epithelial cells. Accessibility peaks and gene expression (violin plots at right) in indicated cell types (see FIG. 3A) are shown for the Nkx3.1 genomic locus, together with position of AR binding site (ARBS) in the 3’ enhancer (Refs. 59, 60).

[0206] To examine the effects of androgen-deprivation on the benign human prostate as well as hormone-sensitive prostate cancer, we have established a small retrospective cohort of prostate samples from prostate cancer patients who received neoadjuvant ADT prior to prostatectomy. This cohort consists of 13 patients with high-risk localized prostate adenocarcinoma who were pre-treated with ADT, together with 13 control patients matched in age and Gleason grade who did not receive pre-treatment. All patients were between the ages of 50-75; in addition, 5 of these patients were enrolled in a clinical trial in which scRNA-seq was performed on their tumor samples. We have performed analyses of several of these patient samples, as shown in FIG. 9A.

[0207] Our studies indicate that the extrinsic pathway for castration-response is mediated by the activity of pleiotrophin (PTN), an AR-regulated signaling factor that is expressed in the prostate stroma. In particular, our findings suggest that PTN signaling activates the mTORCl-SREBPl-SCDl pathway within distal luminal cells to regulate MUFA biosynthesis and repress ferroptosis (FIG. 2).

[0208] Several recent studies have examined potential approaches for inducing ferroptosis as a therapeutic strategy for prostate cancer (Refs 81-84). Our work suggests that such pro-ferroptotic therapies could have greater efficacy if they were to recapitulate the endogenous intrinsic and extrinsic pathways that promote ferroptosis under the physiological contexts that occur during androgen-deprivation. Furthermore, the dual pathway fail-safe mechanism that safeguards against accidental ferroptosis in the absence of androgendeprivation may also ensure that castration-resistance is particularly difficult to overcome in prostate cancer. For example, despite the long-understood role of PI3K-AKT-mT0R signaling in castration-resistance, targeted therapies against this pathway have been relatively ineffective in clinical trials to date (Ref 85).

[0209] References Cited1) Huggins, C. and Hodges, C. V. (1941). The effect of castration, of estrogens, and of androgen injection on serum phosphatase in metastatic carcinoma of prostate. Cancer Res 1, 293-297. aacrjournals.org / cancerres / article- pdf / l / 4 / 293 / 2368295 / crs0010040293.pdf44ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 20252) Shen, M. M. and Abate-Shen, C. (2010). Molecular genetics of prostate cancer: new prospects for old challenges. Genes Dev 24, 1967-2000. PMCID: PMC2939361. pubmed. ncbi.nlm.nih.gov / 20844012 / 3) Toivanen, R. and Shen, M. M. (2017). Prostate organogenesis: tissue induction, hormonal regulation and cell type specification. Development 144, 1382-1398. PMCID: PMC5399670. pubmed. ncbi.nlm.nih.gov / 28400434 / 4) Wang, G., Zhao, D., Spring, D. J. and DePinho, R. A. (2018). Genetics and biology of prostate cancer. Genes Dev 32, 1105-1140. PMCID: PMC6120714. pubmed.ncbi.nlm.nih.gov / 30181359 / 5) Watson, P. A., Arora, V. K. and Sawyers, C. L. (2015). Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. Nat Rev Cancer 15, 701- 711. PMCID: PMC4771416. pubmed.ncbi.nlm.nih.gov / 26563462 / 6) English, H. F., Santen, R. J. and Isaacs, J. T. (1987). Response of glandular versus basal rat ventral prostatic epithelial cells to androgen withdrawal and replacement. Prostate 11, 229-242. PMID: 3684783. pubmed.ncbi.nlm.nih.gov / 3684783 / 7) Evans, G. S. and Chandler, J. A. (1987). Cell proliferation studies in the rat prostate: II.The effects of castration and androgen-induced regeneration upon basal and secretory cell proliferation. Prostate 11, 339-351. PMID: 3684785. pubmed.ncbi.nlm.nih.gov / 3684785 / 8) Kyprianou, N. and Isaacs, J. T. (1988). Activation of programmed cell death in the rat ventral prostate after castration. Endocrinology 122, 552-562. PMID: 2828003. pubmed. ncbi.nlm.nih.gov / 2828003 / 9) Banerjee, S., Banerjee, P. P. and Brown, T. R. (2000). Castration-induced apoptotic cell death in the Brown Norway rat prostate decreases as a function of age. Endocrinology 141, 821-832. PMID: 10650965. pubmed.ncbi.nlm.nih.gov / 10650965 / 10) English, H. F., Kyprianou, N. and Isaacs, J. T. (1989). Relationship between DNA fragmentation and apoptosis in the programmed cell death in the rat prostate following castration. Prostate 15, 233-250. PMID: 2555799. pubmed.ncbi.nlm.nih.gov / 2555799 / 11) Ohlson, N., Wikstrom, P., Stattin, P. and Bergh, A. (2005). Cell proliferation and apoptosis in prostate tumors and adjacent non-malignant prostate tissue in patients at different time-points after castration treatment. Prostate 62, 307-315. PMID: 15389788. pubmed.ncbi.nlm.nih.gov / 15389788 / 45ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202512) Crowley, L. and Shen, M. M. (2022). Heterogeneity and complexity of the prostate epithelium: New findings from single-cell RNA sequencing studies. Cancer Lett 525, 108-114. PMCID: PMC8629925. pubmed.ncbi.nlm.nih.gov / 34728312 / 13) Cunha, G. R., Donjacour, A. A., Cooke, P. S., Mee, S., Bigsby, R. M., Higgins, S. J. and Sugimura, Y. (1987). The endocrinology and developmental biology of the prostate. Endocr Rev 8, 338-362. PMID: 3308446. pubmed.ncbi.nlm.nih.gov / 3308446 / 14) Marker, P. C., Donjacour, A. A., Dahiya, R. and Cunha, G. R. (2003). Hormonal, cellular, and molecular control of prostatic development. Dev Biol 253, 165-174. PMID: 12645922. pubmed.ncbi.nlm.nih.gov / 12645922 / 15) Simanainen, U., Allan, C. M., Lim, P., McPherson, S., Jimenez, M., Zajac, J. D., Davey, R. A. and Handelsman, D. J. (2007). Disruption of prostate epithelial androgen receptor impedes prostate lobe-specific growth and function. Endocrinology 148, 2264-2272. PMID: 17317769. pubmed.ncbi.nlm.nih.gov / 17317769 / 16) Lai, K. P., Yamashita, S., Vitkus, S., Shyr, C. R., Yeh, S. and Chang, C. (2012). Suppressed prostate epithelial development with impaired branching morphogenesis in mice lacking stromal fibromuscular androgen receptor. Mol Endocrinol 26, 52-66. PMCID: PMC3248327. pubmed.ncbi.nlm.nih.gov / 22135068 / 17) Le, V., He, Y., Aldahl, I, Hooker, E., Yu, E. I, Olson, A., Kim, W. K., Lee, D. H., Wong, M., Sheng, R., Mi, J., Geradts, J., Cunha, G. R. and Sun, Z. (2020). Loss of androgen signaling in mesenchymal sonic hedgehog responsive cells diminishes prostate development, growth, and regeneration. PLoS Genet 16, el008588. PMCID: PMC6980684. pubmed. ncbi.nlm.nih.gov / 31929563 / 18) Lee, D. H., Olson, A. W., Wang, J., Kim, W. K., Mi, J., Zeng, H., Le, V., Aldahl, J., Hiroto, A., Wu, X. and Sun, Z. (2021). Androgen action in cell fate and communication during prostate development at single-cell resolution. Development 148, dev 196048. PMCID: PMC7823163. pubmed.ncbi.nlm.nih.gov / 33318148 / 19) Kurita, T., Wang, Y. Z., Donjacour, A. A., Zhao, C., Lydon, J. P., OMalley, B. W ., Isaacs, J. T., Dahiya, R. and Cunha, G. R. (2001). Paracrine regulation of apoptosis by steroid hormones in the male and female reproductive system. Cell Death Differ 8, 192- 200. PMID: 11313721. pubmed. ncbi.nlm.nih.gov / 11313721 / 46ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202520) Dixon, S. J., Lemberg, K. M., Lamprecht, M. R., Skouta, R., Zaitsev, E. M., Gleason, C. E., Patel, D. N., Bauer, A. J., Cantley, A. M., Yang, W. S., Morrison, B., 3rd and Stockwell, B. R. (2012). Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060-1072. PMCID: PMC3367386. pubmed. ncbi.nlm.nih.gov / 22632970 / 21) Jiang, X., Stockwell, B. R. and Conrad, M. (2021). Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol 22, 266-282. PMCID: PMC8142022. pubmed.ncbi.nlm.nih.gov / 33495651 / 22) Stockwell, B. R. (2022). Ferroptosis turns 10: Emerging mechanisms, physiological functions, and therapeutic applications. Cell 185, 2401-2421. PMCID: PMC9273022. pubmed. ncbi.nlm.nih.gov / 35803244 / 23) Stockwell, B. R., Friedmann Angeli, J. P., Bayir, EL, Bush, A. I., Conrad, M., Dixon, S. J., Fulda, S., Gascon, S., Hatzios, S. K., Kagan, V. E., Noel, K., Jiang, X., Linkermann, A., Murphy, M. E., Overholtzer, M., Oyagi, A., Pagnussat, G. C., Park, J., Ran, Q., Rosenfeld, C. S., Salnikow, K., Tang, D., Torti, F. M., Torti, S. V., Toyokuni, S., Woerpel, K. A. and Zhang, D. D. (2017). Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. Cell 171, 273-285. PMCID: PMC5685180. pubmed.ncbi.nlm.nih.gov / 28985560 / 24) Crowley, L., Cambuli, F., Aparicio, L., Shibata, M., Robinson, B. D., Xuan, S., Li, W., Hibshoosh, EL, Loda, M., Rabadan, R. and Shen, M. M. (2020). A single-cell atlas of the mouse and human prostate reveals heterogeneity and conservation of epithelial progenitors. eLife 9, e59465. PMCID: PMC7529463. pubmed.ncbi.nlm.nih.gov / 32915138 / 25) Guo, W., Li, L., He, J., Liu, Z., Han, M., Li, F., Xia, X., Zhang, X., Zhu, Y., Wei, Y.,Li, Y., Aji, R., Dai, H., Wei, H., Li, C., Chen, Y., Chen, L. and Gao, D. (2020). Singlecell transcriptomics identifies a distinct luminal progenitor cell type in distal prostate invagination tips. Nat Genet 52, 908-918. PMCID: PMC8383310. pubmed. ncbi.nlm.nih.gov / 32807988 / 26) Karthaus, W. R., Hofiree, M., Choi, D., Linton, E. L., Turkekul, M., Bejnood, A., Carver, B., Gopalan, A., Abida, W., Laudone, V., Biton, M., Chaudhary, O., Xu, T., Masilionis, L, Manova, K., Mazutis, L., Pe'er, D., Regev, A. and Sawyers, C. L. (2020). Regenerative potential of prostate luminal cells revealed by single-cell analysis. Science 368, 497-505. PMCID: PMC7313621. pubmed. ncbi.nlm.nih.gov / 32355025 / 47ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202527) Joseph, D. B., Henry, G. H., Malewska, A., Iqbal, N. S., Ruetten, H. M., Turco, A. E.,Abler, L. L., Sandhu, S. K., Cadena, M. T., Malladi, V. S., Reese, J. C., Mauck, R. J., Gahan, J. C., Hutchinson, R. C., Roehrborn, C. G., Baker, L. A., Vezina, C. M. and Strand, D. W. (2020). Urethral luminal epithelia are castration-insensitive cells of the proximal prostate. Prostate 80, 872-884. PMCID: PMC7339731. pubmed.ncbi.nlm.nih.gov / 32497356 / 28) Mevel, R., Steiner, I., Mason, S., Galbraith, L. C., Patel, R., Fadlullah, M. Z., Ahmad, I., Leung, H. Y., Oliveira, P., Blyth, K., Baena, E. and Lacaud, G. (2020). RUNX1 marks a luminal castration-resistant lineage established at the onset of prostate development. eLife 9, e60225. PMCID: PMC7644213. pubmed. ncbi.nlm.nih.gov / 33025905 / 29) Henry, G. H., Malewska, A., Joseph, D. B., Malladi, V. S., Lee, J., Torrealba, J., Mauck, R. J., Gahan, J. C., Raj, G. V., Roehrborn, C. G., Hon, G. C., MacConmara, M. P., Reese, J. C., Hutchinson, R. C., Vezina, C. M. and Strand, D. W. (2018). A cellular anatomy of the normal adult human prostate and prostatic urethra. Cell Rep 25, 3530- 3542. PMCID: PMC6411034. pubmed.ncbi.nlm.nih.gov / 30566875 / 30) Tabula Sapiens, C., et al. (2022). The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans. Science 376, eabl4896. PMCID: PMC9812260. pubmed. ncbi.nlm.nih.gov / 35549404 / 31) Aparicio, L., Crowley, L., Christin, J. R., Laplaca, C. J., Hibshoosh, H., Rabadan, R. and Shen, M. M. (2024). Meta-analyses of mouse and human prostate single-cell transcriptomes reveal widespread epithelial plasticity in tissue regression, regeneration, and cancer. Biorxiv doi: 10.1101 / 2024.01.30.578066. PMCID: PMC10862785. pubmed.ncbi.nlm.nih.gov / 38352515 / 32) Bhatia-Gaur, R., Donjacour, A. A., Sciavolino, P. J., Kim, M., Desai, N., Young, P., Norton, C. R., Gridley, T., Cardiff, R. D., Cunha, G. R., Abate-Shen, C. and Shen, M. M. (1999). Roles for Nkx3.1 in prostate development and cancer. Genes Dev 13, 966- 977. PMCID: PMC316645. pubmed.ncbi.nlm.nih.gov / 10215624 / 33) Dutta, A., Le Magnen, C., Mitrofanova, A., Ouyang, X., Califano, A. and Abate-Shen, C. (2016). Identification of an NKX3.1-G9a-UTY transcriptional regulatory network that controls prostate differentiation. Science 352, 1576-1580. PMCID: PMC5507586. pubmed.ncbi.nlm.nih.gov / 27339988 / 34) Abate-Shen, C., Shen, M. M. and Gelmann, E. (2008). Integrating differentiation and cancer: the Nkx3.1 homeobox gene in prostate organogenesis and carcinogenesis.48ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025Differentiation 76, 717-727. PMCID: PMC3683569. pubmed.ncbi.nlm.nih.gov / 18557759 / 35) Kirk, J. S., Wang, J., Long, M., Rosario, S., Tracz, A., Ji, Y., Kumar, R., Liu, X., Jamroze, A., Singh, P. K., Puzanov, I., Chatta, G., Cheng, Q., Huang, J., Wrana, J. L., Lovell, J., Yu, H., Liu, S., Shen, M. M., Liu, T. and Tang, D. G. (2024). Integrated single-cell analysis defines the epigenetic basis of castration-resistant prostate luminal cells. Cell Stem Cell doi: 10.1016 / j.stem.2024.05.008. PMID: 38878775. pubmed.ncbi.nlm.nih.gov / 38878775 / 36) Wu, C. T., Altuwaijri, S., Rieke, W. A., Huang, S. P., Yeh, S., Zhang, C., Niu, Y., Tsai, M. Y. and Chang, C. (2007). Increased prostate cell proliferation and loss of cell differentiation in mice lacking prostate epithelial androgen receptor. Proc Natl Acad Sci USA 104, 12679-12684. PMCID: PMC1937526. pubmed.ncbi.nlm.nih.gov / 17652515 / 37) Zhang, B., Kwon, O. J., Henry, G., Malewska, A., Wei, X., Zhang, L., Brinkley, W., Zhang, Y., Castro, P. D., Titus, M., Chen, R., Sayeeduddin, M., Raj, G. V., Mauck, R., Roehrborn, C., Creighton, C. J., Strand, D. W ., Ittmann, M. M. and Xin, L. (2016). Non-cell-autonomous regulation of prostate epithelial homeostasis by androgen receptor. Mol Cell 63, 976-989. PMCID: PMC5026614. pubmed.ncbi.nlm.nih.gov / 27594448 / 38) Xie, Q., Liu, Y., Cai, T., Horton, C., Stefanson, J. and Wang, Z. A. (2017). Dissecting cell-type-specific roles of androgen receptor in prostate homeostasis and regeneration through lineage tracing. Nat Commun 8, 14284. PMCID: PMC5264212. pubmed.ncbi.nlm.nih.gov / 28112153 / 39) Gao, D., Zhan, Y., Di, W ., Moore, A. R., Sher, J. J., Guan, Y., Wang, S., Zhang, Z., Murphy, D. A., Sawyers, C. L., Chi, P. and Chen, Y. (2016). A Tmprss2-CreERT2 knock-in mouse model for cancer genetic studies on prostate and colon. PLoS One 11, e0161084. PMCID: PMC4990297. pubmed.ncbi.nlm.nih.gov / 27536883 / 40) Doll, S., Proneth, B., Tyurina, Y. Y., Panzilius, E., Kobayashi, S., Ingold, I., Irmler, M., Beckers, J., Aichler, M., Walch, A., Prokisch, H., Trumbach, D., Mao, G., Qu, F., Bayir, H., Fullekrug, J., Scheel, C. H., Wurst, W ., Schick, J. A., Kagan, V. E., Angeli, J. P. and Conrad, M. (2017). ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. Nat Chem Biol 13, 91-98. PMCID: PMC5610546. pubmed. ncbi.nlm.nih.gov / 27842070 / 49ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202541) Tummers, B. and Green, D. R. (2017). Caspase-8: regulating life and death. Immunol Rev 277, 76-89. PMCID: PMC5417704. pubmed.ncbi.nlm.nih.gov / 28462525 / 42) He, W. T., Wan, H., Hu, L., Chen, P., Wang, X., Huang, Z., Yang, Z. H., Zhong, C. Q. and Han, J. (2015). Gasdermin D is an executor of pyroptosis and required for interleukin-lbeta secretion. Cell Res 25, 1285-1298. PMCID: PMC4670995. pubmed.ncbi.nlm.nih.gov / 26611636 / 43) Shi, J., Zhao, Y., Wang, K., Shi, X., Wang, Y., Huang, H., Zhuang, Y., Cai, T., Wang, F. and Shao, F. (2015). Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660-665. PMID: 26375003. pubmed. ncbi.nlm.nih.gov / 26375003 / 44) Feng, H., Schorpp, K., Jin, J., Yozwiak, C. E., Hoffstrom, B. G., Decker, A. M., Rajbhandari, P., Stokes, M. E., Bender, H. G., Csuka, J. M., Upadhyayula, P. S., Canoil, P., Uchida, K., Soni, R. K., Hadian, K. and Stockwell, B. R. (2020). Transferrin receptor is a specific ferroptosis marker. Cell Rep Q, 3411-3423 e3417. PMCID: PMC7172030. pubmed.ncbi.nlm.nih.gov / 32160546 / 45) Nelson, P. S., Clegg, N., Arnold, H., Ferguson, C., Bonham, M., White, J., Hood, L. and Lin, B. (2002). The program of androgen-responsive genes in neoplastic prostate epithelium. Proc Natl Acad Set USA 99, 11890-11895. PMCID: PMC129364. pubmed.ncbi.nlm.nih.gov / 12185249 / 46) Bieberich, C. J., Fujita, K., He, W. W. and Jay, G. (1996). Prostate-specific and androgen-dependent expression of a novel homeobox gene. J. Biol. Chem. 271, 31779- 31782. PMID: 8943214. pubmed.ncbi.nlm.nih.gov / 8943214 / 47) Tan, P. Y., Chang, C. W ., Chng, K. R., Wansa, K. D., Sung, W. K. and Cheung, E. (2012). Integration of regulatory networks by NKX3-1 promotes androgen-dependent prostate cancer survival. Mol Cell Biol 32, 399-414. PMCID: PMC3255774. pubmed. ncbi.nlm.nih.gov / 22083957 / 48) Ouyang, X., DeWeese, T. L., Nelson, W. G. and Abate-Shen, C. (2005). Loss-of- function of Nkx3.1 promotes increased oxidative damage in prostate carcinogenesis. Cancer Res 65, 6773-6779. PMID: 16061659. pubmed.ncbi.nlm.nih.gov / 16061659 / 49) Papachristodoulou, A., Rodriguez-Calero, A., Panja, S., Margolskee, E., Virk, R. K., Milner, T. A., Pina Martina, L., Kim, J. Y., Di Bernardo, M., Williams, A. B., Maliza, E. A., Caputo, J. M., Haas, C., Wang, V., De Castro, G. J., Wenske, S., Hibshoosh, H., McKiernan, J. M., Shen, M. M., Rubin, M. A., Mitrofanova, A., Dutta, A. and Abate-50ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025Shen, C. (2021). NKX3.1 localization to mitochondria suppresses prostate cancer initiation. Cancer Discov 11 : 2316-2333. PMCID: PMC7611624. pubmed.ncbi.nlm.nih.gov / 33893149 / 50) Orr, B., Vanpoucke, G., Grace, O. C., Smith, L., Anderson, R. A., Riddick, A. C., Franco, O. E., Hayward, S. W. and Thomson, A. A. (2011). Expression of pleiotrophin in the prostate is androgen regulated and it functions as an autocrine regulator of mesenchyme and cancer associated fibroblasts and as a paracrine regulator of epithelia. Prostate 71, 305-317. PMCID: PMC3045659. pubmed. ncbi.nlm.nih.gov / 20812209 / 51) Yi, J., Zhu, J., Wu, J., Thompson, C. B. and Jiang, X. (2020). Oncogenic activation of PI3K-AKT-mT0R signaling suppresses ferroptosis via SREBP-mediated lipogenesis. Proc Natl Acad Set USA 117, 31189-31197. PMCID: PMC7733797. pubmed.ncbi.nlm.nih.gov / 33229547 / 52) Shen, M. M. and Abate-Shen, C. (2007). Pten inactivation and the emergence of androgen-independent prostate cancer. Cancer Res 67, 6535-6538. PMID: 17638861. pubmed.ncbi.nlm.nih.gov / 17638861 / 53) Wang, S., Gao, J., Lei, Q., Rozengurt, N., Pritchard, C., Jiao, J., Thomas, G. V., Li, G., Roy-Burman, P., Nelson, P. S., Liu, X. and Wu, H. (2003). Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell 4, 209-221. PMID: 14522255. pubmed.ncbi.nlm.nih.gov / 14522255 / 54) Alumkal, J. J., Sun, D., Lu, E., Beer, T. M., Thomas, G. V., Latour, E., Aggarwal, R., Cetnar, J., Ryan, C. J., Tabatabaei, S., Bailey, S., Turina, C. B., Quigley, D. A., Guan, X., Foye, A., Youngren, J. F., Urrutia, J., Huang, J., Weinstein, A. S., Friedl, V., Rettig, M., Reiter, R. E., Spratt, D. E., Gleave, M., Evans, C. P., Stuart, J. M., Chen, Y., Feng, F. Y., Small, E. J., Witte, O. N. and Xia, Z. (2020). Transcriptional profiling identifies an androgen receptor activity-low, sternness program associated with enzalutamide resistance. Proc Natl Acad Set USA 117, 12315-12323. PMCID: PMC7275746. pubmed.ncbi.nlm.nih.gov / 32424106 / 55) Kim, R., Hashimoto, A., Markosyan, N., Tyurin, V. A., Tyurina, Y. Y ., Kar, G., Fu, S., Sehgal, M., Garcia-Gerique, L., Kossenkov, A., Gebregziabher, B. A., Tobias, J. W., Hicks, K., Halpin, R. A., Cvetesic, N., Deng, H., Donthireddy, L., Greenberg, A., Nam, B., Vonderheide, R. H., Nefedova, Y., Kagan, V. E. and Gabrilovich, D. I. (2022). Ferroptosis of tumour neutrophils causes immune suppression in cancer. Nature 612, 338-346. PMCID: PMC9875862. pubmed.ncbi.nlm.nih.gov / 36385526 / 51ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202556) Liberzon, A., Birger, C., Thorvaldsdottir, H., Ghandi, M., Mesirov, J. P. and Tamayo, P. (2015). The Molecular Signatures Database (MSigDB) hallmark gene set collection. CellSyst 1, 417-425. PMCID: PMC4707969. pubmed.ncbi.nlm.nih.gov / 26771021 / 57) Schiebinger, G., Shu, J., Tabaka, M., Cleary, B., Subramanian, V., Solomon, A., Gould, J., Liu, S., Lin, S., Berube, P., Lee, L., Chen, J., Brumbaugh, J., Rigollet, P., Hochedlinger, K., Jaenisch, R., Regev, A. and Lander, E. S. (2019). Optimal-transport analysis of single-cell gene expression identifies developmental trajectories in reprogramming. Cell 176, 928-943. PMCID: PMC6402800. pubmed. ncbi.nlm.nih.gov / 30712874 / 58) Liang, D., Feng, Y., Zandkarimi, F., Wang, EL, Zhang, Z., Kim, J., Cai, Y., Gu, W Stockwell, B. R. and Jiang, X. (2023). Ferroptosis surveillance independent of GPX4 and differentially regulated by sex hormones. Cell 186, 2748-2764 e2722. PMCID: PMC10330611. pubmed.ncbi.nlm.nih.gov / 37267948 / 59) Chen, EL, Mutton, L. N., Prins, G. S. and Bieberich, C. J. (2005). Distinct regulatory elements mediate the dynamic expression pattern of Nkx3.1. Dev Dyn 234, 961-973. PMCID: PMC2819389. pubmed.ncbi.nlm.nih.gov / 16245334 / 60) Xie, Q. and Wang, Z. A. (2017). Transcriptional regulation of the Nkx3.1 gene in prostate luminal stem cell specification and cancer initiation via its 3' genomic region. JBiol Chem 292, 13521-13530. PMCID: PMC5566512. pubmed.ncbi.nlm.nih.gov / 28679531 / 61) Kaya-Okur, H. S., Janssens, D. EL, Henikoff, J. G., Ahmad, K. and Henikoff, S. (2020). Efficient low-cost chromatin profiling with CUT&Tag. Nat Protoc 15, 3264-3283. PMCID: PMC8318778. pubmed.ncbi.nlm.nih.gov / 32913232 / 62) Kaya-Okur, H. S., Wu, S. J., Codomo, C. A., Pledger, E. S., Bryson, T. D., Henikoff, J. G., Ahmad, K. and Henikoff, S. (2019). CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10, 1930. PMCID: PMC6488672. pubmed. ncbi.nlm.nih.gov / 31036827 / 63) Li, J. J., Vasciaveo, A., Karagiannis, D., Sun, Z., Chen, X., Socciarelli, F., Frankenstein, Z., Zou, M., Pannellini, T., Chen, Y., Gardner, K., Robinson, B. D., de Bono, J., Abate- Shen, C., Rubin, M. A., Loda, M., Sawyers, C. L., Califano, A., Lu, C. and Shen, M. M. (2023). NSD2 maintains lineage plasticity and castration-resistance in neuroendocrine prostate cancer. bioRxiv doi: 10.1101 / 2023.07.18.549585. PMCID: PMC 10370123. pubmed. ncbi.nlm.nih.gov / 37502956 / 52ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202564) Tian, H., Rajbhandari, P., Tarolli, J., Decker, A. M., Neelakantan, T. V., Angerer, T., Zandkarimi, F., Remotti, H., Frache, G., Winograd, N. and Stockwell, B. R. (2024). Multimodal mass spectrometry imaging identifies cell-type-specific metabolic and lipidomic variation in the mammalian liver. Dev Cell 59, 869-881 e866. PMID: 38359832. pubmed.ncbi.nlm.nih.gov / 38359832 / 65) Zhang, Y., Tan, H., Daniels, J. D., Zandkarimi, F., Liu, H., Brown, L. M., Uchida, K., O'Connor, O. A. and Stockwell, B. R. (2019). Imidazole ketone erastin induces ferroptosis and slows tumor growth in a mouse lymphoma model. Cell Chem Biol 26, 623-633 e629. PMCID: PMC6525071. pubmed.ncbi.nlm.nih.gov / 30799221 / 66) Upadhyayula, P. S., Higgins, D. M., Mela, A., Banu, M., Dovas, A., Zandkarimi, F., Patel, P., Mahajan, A., Humala, N., Nguyen, T. T. T., Chaudhary, K. R., Liao, L., Argenziano, M., Sudhakar, T., Sperring, C. P., Shapiro, B. L., Ahmed, E. R., Kinslow, C., Ye, L. F., Siegelin, M. D., Cheng, S., Soni, R., Bruce, J. N., Stockwell, B. R. and Canoil, P. (2023). Dietary restriction of cysteine and methionine sensitizes gliomas to ferroptosis and induces alterations in energetic metabolism. Nat Commun 14, 1187. PMCID: PMC9981683. pubmed.ncbi.nlm.nih.gov / 36864031 / 67) Orlando, D. A., Chen, M. W ., Brown, V. E., Solanki, S., Choi, Y. J., Olson, E. R., Fritz, C. C., Bradner, J. E. and Guenther, M. G. (2014). Quantitative ChlP-Seq normalization reveals global modulation of the epigenome. Cell Rep 9, 1163-1170. PMID: 25437568. pubmed.ncbi.nlm.nih.gov / 25437568 / 68) Janssens, D. H., Greene, J. E., Wu, S. J., Codomo, C. A., Minot, S. S., Furlan, S. N., Ahmad, K. and Henikoff, S. (2024). Scalable single-cell profiling of chromatin modifications with sciCUT&Tag. Nat Protoc 19, 83-112. PMCID: PMC11229882. pubmed.ncbi.nlm.nih.gov / 37935964 / 69) Barcenas-Walls, J. R., Ansaloni, F., Herve, B., Strandback, E., Nyman, T., Castelo- Branco, G. and Bartosovic, M. (2024). Nano-CUT&Tag for multimodal chromatin profiling at single-cell resolution. Nat Protoc 19, 791-830. PMID: 38129675. pubmed.ncbi.nlm.nih.gov / 38129675 / 70) Karlsson, M., Zhang, C., Mear, L., Zhong, W., Digre, A., Katona, B., Sjostedt, E., Butler, L., Odeberg, J., Dusart, P., Edfors, F., Oksvold, P., von Feilitzen, K., Zwahlen, M., Arif, M., Altay, O., Li, X., Ozcan, M., Mardinoglu, A., Fagerberg, L., Mulder, J., Luo, Y., Ponten, F., Uhlen, M. and Lindskog, C. (2021). A single-cell type transcriptomics map of human tissues. Sci Adv 7, eabh2169. PMCID: PMC8318366. pubmed.ncbi.nlm.nih.gov / 34321199 / 53ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202571) Uhlen, M., Fagerberg, L., Hallstrom, B. M., Lindskog, C., Oksvold, P., Mardinoglu, A., Sivertsson, A., Kampf, C., Sjostedt, E., Asplund, A., Olsson, I., Edlund, K., Lundberg, E., Navani, S., Szigyarto, C. A., Odeberg, J., Djureinovic, D., Takanen, J. O., Hober, S., Alm, T., Edqvist, P. H., Berling, H., Tegel, H., Mulder, J., Rockberg, J., Nilsson, P., Schwenk, J. M., Hamsten, M., von Feilitzen, K., Forsberg, M., Persson, L., Johansson, F., Zwahlen, M., von Heijne, G., Nielsen, J. and Ponten, F. (2015). Proteomics. Tissue-based map of the human proteome. Science 347, 1260419. PMID: 25613900. pubmed. ncbi.nlm.nih.gov / 25613900 / 72) Pakula, H., Omar, M., Carelli, R., Pederzoli, F., Fanelli, G. N., Pannellini, T., Socciarelli, F., Van Emmenis, L., Rodrigues, S., Fidalgo-Ribeiro, C., Nuzzo, P. V., Brady, N. J., Dinalankara, W., Jere, M., Valencia, I., Saladino, C., Stone, J., Unkenholz, C., Gamer, R., Alexanderani, M. K., Khani, F., de Almeida, F. N., Abate-Shen, C., Greenblatt, M. B., Rickman, D. S., Barbieri, C. E., Robinson, B. D., Marchionni, L. and Loda, M. (2024). Distinct mesenchymal cell states mediate prostate cancer progression. Nat Commun 15, 363. PMCID: PMC10774315. pubmed.ncbi.nlm.nih.gov / 38191471 / 73) Xu, C., Zhu, S., Wu, M., Han, W. and Yu, Y. (2014). Functional receptors and intracellular signal pathways of midkine (MK) and pleiotrophin (PTN). Biol Pharm Bull 37, 511-520. PMID: 24694599. pubmed.ncbi.nlm.nih.gov / 24694599 / 74) Harroch, S., Palmeri, M., Rosenbluth, J., Custer, A., Okigaki, M., Shrager, P., Blum, M., Buxbaum, J. D. and Schlessinger, J. (2000). No obvious abnormality in mice deficient in receptor protein tyrosine phosphatase beta. Mol Cell Biol 20, 7706-7715. PMCID: PMC86347. pubmed. ncbi.nlm.nih.gov / 11003666 / 75) Lafont, D., Adage, T., Greco, B. and Zaratin, P. (2009). A novel role for receptor like protein tyrosine phosphatase zeta in modulation of sensorimotor responses to noxious stimuli: evidences from knockout mice studies. Behav Brain Res 201, 29-40. PMID: 19428613. pubmed. ncbi .nlm.nih.gov / 19428613 / 76) Miyazaki, M., Flowers, M. T., Sampath, H., Chu, K., Otzelberger, C., Liu, X. and Ntambi, J. M. (2007). Hepatic stearoyl-CoA desaturase-1 deficiency protects mice from carbohydrate-induced adiposity and hepatic steatosis. Cell Metab 6, 484-496. PMID: 18054317. pubmed. ncbi. nlm.nih.gov / 18054317 / 77) Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M. and Altman, D. G. (2010). Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoSBiol , el000412. PMCID: PMC2893951. pubmed.ncbi.nlm.nih.gov / 20613859 / 54ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202578) Minas, T. Z., Candia, J., Dorsey, T. H., Baker, F., Tang, W., Kiely, M., Smith, C. J., Zhang, A. L., Jordan, S. V., Obadi, O. M., Ajao, A., Tettey, Y., Biritwum, R. B., Adjei, A. A., Mensah, J. E., Hoover, R. N., Jenkins, F. J., Kittles, R., Hsing, A. W., Wang, X. W., Loffredo, C. A., Yates, C., Cook, M. B. and Ambs, S. (2022). Serum proteomics links suppression of tumor immunity to ancestry and lethal prostate cancer. Nat Commun 13, 1759. PMCID: PMC8975871. pubmed.ncbi.nlm.nih.gov / 35365620 / 79) Bilsland, J. G., Wheeldon, A., Mead, A., Znamenskiy, P., Almond, S., Waters, K. A., Thakur, M., Beaumont, V., Bonnert, T. P., Heavens, R., Whiting, P., McAllister, G. and Munoz- Sanjuan, I. (2008). Behavioral and neurochemical alterations in mice deficient in anaplastic lymphoma kinase suggest therapeutic potential for psychiatric indications. Neuropsychopharmacology 33, 685-700. PMID: 17487225. pubmed.ncbi.nlm.nih.gov / 17487225 / 80) Weiss, J. B., Xue, C., Benice, T., Xue, L., Morris, S. W. and Raber, J. (2012). Anaplastic lymphoma kinase and leukocyte tyrosine kinase: functions and genetic interactions in learning, memory and adult neurogenesis. Pharmacol Biochem Behav 100, 566-574. PMID: 22079349. pubmed. ncbi.nlm.nih.gov / 22079349 / 81) Cao, P. H. A., Dominic, A., Lujan, F. E., Senthilkumar, S., Bhattacharya, P. K., Frigo, D. E. and Subramani, E. (2024). Unlocking ferroptosis in prostate cancer - the road to novel therapies and imaging markers. Nat Rev Urol doi: 10.1038 / s41585-024-00869-9. PMID: 38627553.82) Ghoochani, A., Hsu, E. C., Aslan, M., Rice, M. A., Nguyen, H. M., Brooks, J. D., Corey, E., Paulmurugan, R. and Stoyanova, T. (2021). Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer. Cancer Res 81, 1583-1594. PMCID: PMC7969452. pubmed.ncbi.nlm.nih.gov / 33483372 / 83) Sun, R., Yan, B., Li, H., Ding, D., Wang, L., Pang, J., Ye, D. and Huang, H. (2023). Androgen receptor variants confer castration resistance in prostate cancer by counteracting antiandrogen-induced ferroptosis. Cancer Res 83, 3192-3204. PMCID: PMC10543964. pubmed.ncbi.nlm.nih.gov / 37527336 / 84) Wang, M. E., Chen, J., Lu, Y., Bawcom, A. R., Wu, J., Ou, J., Asara, J. M., Armstrong, A. J., Wang, Q., Li, L., Wang, Y., Huang, J. and Chen, M. (2023). RB1- deficient prostate tumor growth and metastasis are vulnerable to ferroptosis induction55ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 via the E2F / ACSL4 axis. J Clin Invest 133, PMCID: PMC10178842. pubmed.ncbi.nlm.nih.gov / 36928314 / 85) Wylaz, M., Kaczmarska, A., Pajor, D., Hryniewicki, M., Gil, D. and Dulinska-Litewka, J. (2023). Exploring the role of PI3K / AKT / mT0R inhibitors in hormone-related cancers: A focus on breast and prostate cancer. Biomed Pharmacother 168, 115676. PMID: 37832401. pubmed. ncbi.nlm.nih.gov / 37832401 / 86) Maund, S. L., Nolley, R. and Peehl, D. M. (2014). Optimization and comprehensive characterization of a faithful tissue culture model of the benign and malignant human prostate. Lab Invest 94, 208-221. PMCID: PMC3946793. pubmed.ncbi.nlm.nih.gov / 24296879 / 87) Perez, L. M. and Nonn, L. (2022). Harnessing the utility of ex vivo patient prostate tissue slice cultures. Front Oncol 12, 864723. PMCID: PMC9008363. pubmed.ncbi.nlm.nih.gov / 35433436 / 88) Liu, H., Forouhar, F., Lin, A. J., Wang, Q., Polychronidou, V., Soni, R. K., Xia, X. and Stockwell, B. R. (2022). Small-molecule allosteric inhibitors of GPX4. Cell Chem Biol 29, 1680-1693 el689. PMCID: PMC9772252. pubmed.ncbi.nlm.nih.gov / 36423641 / 89) Yang, W. S., SriRamaratnam, R., Welsch, M. E., Shimada, K., Skouta, R., Viswanathan, V. S., Cheah, J. H., Clemons, P. A., Shamji, A. F., Clish, C. B., Brown, L. M., Girotti, A. W ., Cornish, V. W ., Schreiber, S. L. and Stockwell, B. R. (2014). Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317-331. PMCID: PMC4076414. pubmed.ncbi.nlm.nih.gov / 24439385 / 90) Bailey, H. H., Mulcahy, R. T., Tutsch, K. D., Arzoomanian, R. Z., Alberti, D., Tombes, M. B., Wilding, G., Pomplun, M. and Spriggs, D. R. (1994). Phase I clinical trial of intravenous L-buthionine sulfoximine and melphalan: an attempt at modulation of glutathione. J Clin Oncol 12, 194-205. PMID: 8270977. pubmed. ncbi.nlm.nih.gov / 8270977 / 91) lanevski, A., Giri, A. K. and Aittokallio, T. (2022). SynergyFinder 3.0: an interactive analysis and consensus interpretation of multi-drug synergies across multiple samples. Nucleic Acids Res 50, W739-W743. PMCID: PMC9252834. pubmed. ncbi.nlm.nih.gov / 35580060 / 92) Ellwood-Yen, K., Graeber, T. G., Wongvipat, J., Iruela-Arispe, M. L., Zhang, J., Matusik, R., Thomas, G. V. and Sawyers, C. L. (2003). Myc-driven murine prostate56ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 cancer shares molecular features with human prostate tumors. Cancer Cell 4, 223-238. PMID: 14522256. pubmed.ncbi.nlm.nih.gov / 14522256 / 93) Zou, M., Toivanen, R., Mitrofanova, A., Floch, N., Hayati, S., Sun, Y ., Le Magnen, C., Chester, D., Mostaghel, E. A., Califano, A., Rubin, M. A., Shen, M. M. and Abate- Shen, C. (2017). Transdifferentiation as a mechanism of treatment resistance in a mouse model of castration-resistant prostate cancer. Cancer Discov 7, 736-749. PMCID: PMC5501744. pubmed.ncbi.nlm.nih.gov / 28411207 / 94) Corey, E., Quinn, J. E., Buhler, K. R., Nelson, P. S., Macoska, J. A., True, L. D. and Vessella, R. L. (2003). LuCaP 35: a new model of prostate cancer progression to androgen independence. Prostate 55, 239-246. PMID: 12712403. pubmed. ncbi.nlm.nih.gov / 12712403 / 95) Nguyen, H. M., Vessella, R. L., Morrissey, C., Brown, L. G., Coleman, I. M., Higano, C. S., Mostaghel, E. A., Zhang, X., True, L. D., Lam, H. M., Roudier, M., Lange, P. H., Nelson, P. S. and Corey, E. (2017). LuCaP prostate cancer patient-derived xenografts reflect the molecular heterogeneity of advanced disease an— d serve as models for evaluating cancer therapeutics. Prostate 11, 654-671. PMCID: PMC5354949. pubmed. ncbi.nlm.nih.gov / 28156002 / 96) Vasciaveo, A., Arriaga, J. M., de Almeida, F. N., Zou, M., Douglass, E. F., Picech, F., Shibata, M., Rodriguez-Calero, A., de Brot, S., Mitrofanova, A., Chua, C. W ., Karan, C., Realubit, R., Pampou, S., Kim, J. Y., Afari, S. N., Mukhammadov, T., Zanella, L., Corey, E., Alvarez, M. J., Rubin, M. A., Shen, M. M., Califano, A. and Abate-Shen, C. (2023). OncoLoop: A network-based precision cancer medicine framework. Cancer Discov 13, 386-409. PMCID: PMC9905319. pubmed.ncbi.nlm.nih.gov / 36374194 / 97) Hadian, K., & Stockwell, B. R. (2020). SnapShot: Ferroptosis. Cell, 181(5), 1188— 1188. el. doi.org / 10.1016 / j. cell.2020.04.03957ACTIVEUS 211605386v.l

Claims

Attorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025CLAIMSWhat is claimed is:

1. A method of treating or preventing prostate cancer in a subject in need thereof, comprising modulating the expression of a first component of an intrinsic androgen signaling pathway.

2. The method of claim 1, further comprising modulating the expression of a second component of an extrinsic androgen signaling pathway.

3. The method of claims 1 or 2, wherein the prostate cancer is an early-stage prostate cancer, a Stage 0 prostate cancer, a Stage I prostate cancer, a Stage II prostate cancer, a Stage III prostate cancer, a Stage IV prostate cancer, an advanced stage, or an endstage cancer.

4. The method of any one of claims 1-3, wherein the prostate cancer is castrationresistant prostate cancer (CRPC).

5. The method of any one of claims 1-3, wherein the prostate cancer is a neuroendocrine prostate cancer (NEPC).

6. The method of any one of claims 1-5, wherein the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE).

7. The method of any one of claims 1-6, wherein the prostate cancer is CPRC with androgen receptor expression (CRPC-AR).

8. The method of any one of claim 1-6, wherein the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

9. The method of any one of claims 1-8, wherein the extrinsic androgen signaling pathway occurs at least partially in a stromal microenvironment.

10. The method of any one of claims 1-9, wherein the extrinsic androgen signaling pathway occurs partially in a stromal microenvironment and partially within a prostate luminal epithelial cell.58ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202511. The method of any one of claims 1-10, wherein the intrinsic androgen signaling pathway occurs at least partially within a prostate luminal epithelial cell.

12. The method of any one of claims 1-11, wherein the intrinsic androgen signaling pathway occurs within a prostate luminal epithelial cell.

13. The method of any one of claims 1-12, wherein the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises inducing ferroptosis by downregulating or inhibiting the intrinsic androgen signaling pathway.

14. The method of any one of claims 1-13, wherein the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises inducing lipid peroxidation by downregulating or inhibiting the intrinsic androgen signaling pathway.

15. The method of any one of claims 1-14, wherein the modulating the expression of a first component of an intrinsic androgen signaling pathway comprises disrupting the balance of Monounsaturated Fatty Acids / Polyunsaturated Fatty Acids (MUFA / PUFA) by downregulating or inhibiting the intrinsic androgen signaling pathway.

16. The method of any one of claims 1-15, wherein the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises inducing ferroptosis by downregulating or inhibiting the extrinsic androgen signaling pathway.

17. The method of any one of claims 1-16, wherein the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises inducing lipid peroxidation by downregulating or inhibiting the extrinsic androgen signaling pathway.

18. The method of any one of claims 1-16, wherein the modulating the expression of a second component of an extrinsic androgen signaling pathway comprises disrupting the balance of Monounsaturated Fatty Acids / Polyunsaturated Fatty Acids (MUFA / PUFA) by downregulating or inhibiting the extrinsic androgen signaling pathway.

19. The method of any one of claims 1-18, wherein the first component is NKX3.1, FASN, ACSL3, MBOAT2, or GPX4.59ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202520. The method of any one of claims 1-19, wherein the second component is pleiotrophin (PTN), mTOR, mTORCl, RPTOR, SREBP1, or SCD1.

21. A method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a NKX3.1 inhibitor.

22. The method of claims 21, wherein the prostate cancer is an early-stage prostate cancer, a Stage 0 prostate cancer, a Stage I prostate cancer, a Stage II prostate cancer, a Stage III prostate cancer, a Stage IV prostate cancer, an advanced stage or an endstage cancer.

23. The method of claims 21 or 22, wherein the prostate cancer is castration-resistant prostate cancer (CRPC).

24. The method of any one of claims 21-23, wherein the prostate cancer is a neuroendocrine prostate cancer (NEPC).

25. The method of any one of claims 21-24, wherein the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE).

26. The method of any one of claims 21-25, wherein the prostate cancer is CPRC with androgen receptor expression (CRPC-AR).

27. The method of any one of claim 21-26, wherein the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

28. The method of any one of claims 21-27, wherein the NKX3.1 inhibitor is administered in combination with another NKX3.1 inhibitor or one or more NKX3.1 activity modulator.

29. The method of any one of claims 21-28, wherein the NKX3.1 inhibitor comprises a composition comprising an inhibitory RNA for a messenger RNA sequence encoding a NKX3.1 protein.

30. The method of any one of claims 21-29, wherein the NKX3.1 inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding NKX3.1 gene or controlling transcription of NKX3.1 gene.60ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202531. The method of any one of claims 21-28, wherein the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment.

32. The method of claim 31, wherein the anti-androgen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide.

33. The method of claim 31, wherein the anti-androgen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

34. The method of any one of claims 21-33, wherein the pharmaceutical composition is administered before, after, or in combination with radiation therapy.

35. The method of any one of claims 31-34, wherein the administration of the NKX3.1 inhibitor to the subject in combination with an anti-androgen treatment decreases tumor size.

36. The method of any one of claims 31-35, wherein the administration of the NKX3.1 inhibitor to the subject in combination with an anti-androgen treatment decreases tumor number.

37. The method of any one of claims 31-36, wherein the administration of the NKX3.1 inhibitor to the subject in combination with an anti-androgen treatment prevents cancer metastasis.

38. A method of treating or preventing prostate cancer in a subject in need thereof, the method comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a PTN inhibitor.

39. The method of claims 38, wherein the prostate cancer is an early-stage prostate cancer, a Stage 0 prostate cancer, a Stage I prostate cancer, a Stage II prostate cancer, a Stage III prostate cancer, a Stage IV prostate cancer, an advanced stage or an endstage cancer.

40. The method of claims 38 or 39, wherein the prostate cancer is castration-resistant prostate cancer (CRPC).61ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202541. The method of any one of claims 38-40, wherein the prostate cancer is a neuroendocrine prostate cancer (NEPC).

42. The method of any one of claims 38-41, wherein the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE).

43. The method of any one of claims 38-42, wherein the prostate cancer is CPRC with androgen receptor expression (CRPC-AR).

44. The method of any one of claim 38-43, wherein the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

45. The method of any one of claims 38-44, wherein the PTN inhibitor is administered in combination with another PTN inhibitor or one or more PTN activity modulator.

46. The method of any one of claims 38-45, wherein the PTN inhibitor comprises a composition comprising a small interfering RNA specific for a messenger RNA sequence encoding a PTN protein.

47. The method of any one of claims 38-46, wherein the PTN inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a PTN gene or controlling transcription of a PTN gene.

48. The method of any one of claims 38-47, wherein the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment.

49. The method of claim 48, wherein the anti-androgen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide.

50. The method of claim 48, wherein the anti-androgen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

51. The method of any one of claims 38-50, wherein the pharmaceutical composition is administered before, after, or in combination with radiation therapy.62ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202552. The method of any one of claims 48-51, wherein the administration of the PTN inhibitor to the subject in combination with an anti-androgen treatment decreases tumor size.

53. The method of any one of claims 48-52, wherein the administration of the PTN inhibitor to the subject in combination with an anti-androgen treatment decreases tumor number.

54. The method of any one of claims 48-53, wherein the administration of the PTN inhibitor to the subject in combination with an anti-androgen treatment prevents cancer metastasis.

55. A method of treating or preventing prostate cancer in a subject in need thereof, comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a first inhibitor and a therapeutically effective amount of a second inhibitor, wherein the first inhibitor modulates the expression of a component of an intrinsic androgen signaling pathway and the second inhibitor modulates the expression of a component of an extrinsic androgen signaling pathway.

56. The method of claim 55, wherein the prostate cancer is an early-stage prostate cancer, a Stage 0 prostate cancer, a Stage I prostate cancer, a Stage II prostate cancer, a Stage III prostate cancer, a Stage IV prostate cancer, an advanced stage, or an end-stage cancer.

57. The method of claims 55 or 56, wherein the prostate cancer is castration-resistant prostate cancer (CRPC).

58. The method of claims 55 or 56, wherein the prostate cancer is a neuroendocrine prostate cancer (NEPC).

59. The method of any one of claims 55-58, wherein the prostate cancer is neuroendocrine castration resistant prostate cancer (CRPC-NE).

60. The method of any one of claims 55-59, wherein the prostate cancer is CPRC with androgen receptor expression (CRPC-AR).63ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202561. The method of any one of claim 55-59, wherein the CRPC lacks androgen receptor expression or sensitivity to one or more androgen receptor inhibitors.

62. The method of any one of claims 55-61, wherein the extrinsic androgen signaling pathway occurs at least partially in a stromal microenvironment.

63. The method of any one of claims 55-62, wherein the extrinsic androgen signaling pathway occurs partially in a stromal microenvironment and partially within a prostate luminal epithelial cell.

64. The method of any one of claims 55-63, wherein the intrinsic androgen signaling pathway occurs at least partially within a prostate luminal epithelial cell.

65. The method of any one of claims 55-64, wherein the intrinsic androgen signaling pathway occurs within a prostate luminal epithelial cell.

66. The method of any one of claims 55-65, wherein the first inhibitor comprises a NKX3.1 inhibitor, a FASN inhibitor, a ACSL3 inhibitor, a MBOAT2 inhibitor, GPX4 inhibitor, or any combination thereof.

67. The method of any one of claims 55-66, wherein the first inhibitor comprises a composition comprising an inhibitory RNA for a messenger RNA sequence encoding a NKX3.1 protein, an inhibitory RNA for a messenger RNA sequence encoding a FASN protein, an inhibitory RNA for a messenger RNA sequence encoding a ACSL3 protein, an inhibitory RNA for a messenger RNA sequence encoding a MBOAT2 protein, an inhibitory RNA for a messenger RNA sequence encoding a GPX4 protein, or any combination thereof.

68. The method of any one of claims 55-67, wherein the first inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding NKX3.1 gene or controlling transcription of NKX3.1 gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a FASN gene or controlling transcription of a FASN gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a ACSL3 gene or controlling transcription of a ACSL3 gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene64ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 sequence encoding &MBOAT2 gene or controlling transcription of &MB0AT2 gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a GPX4 gene or controlling transcription of a GPX4 gene, or any combination thereof.

69. The method of any one of claims 55-68, wherein the second inhibitor comprises a PTN inhibitor, a MTOR inhibitor, a mTORCl inhibitor, a RPTOR inhibitor, a SREBP1 inhibitor, a SCD1 inhibitor, or any combination thereof.

70. The method of any one of claims 55-69, wherein the second inhibitor comprises a composition comprising an inhibitory RNA for a messenger RNA sequence encoding a PTN protein, an inhibitory RNA for a messenger RNA sequence encoding a mTORCl protein, an inhibitory RNA for a messenger RNA sequence encoding a MTOR protein, an inhibitory RNA for a messenger RNA sequence encoding a RPTOR protein, an inhibitory RNA for a messenger RNA sequence encoding a SREBP1 protein, an inhibitory RNA for a messenger RNA sequence encoding a SCD1 protein, or any combination thereof.

71. The method of any one of claims 55-70, wherein the second inhibitor comprises a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a PTN gene or controlling transcription of a PTN gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a MTOR gene or controlling transcription of a MTOR gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a mTORCl gene or controlling transcription of a mTORCl gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a RPTOR gene or controlling transcription of a RPTOR gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a SREBP1 gene or controlling transcription of a SREBP1 gene, a CRISPR cassette comprising a gRNA or sgRNA specific for the nucleotide gene sequence encoding a SCD1 gene or controlling transcription of a SCD1 gene, or any combination thereof.

72. The method of any one of claims 55-71, wherein the first inhibitor comprises a NKX3.1 inhibitor, a FASN inhibitor, a ACSL3 inhibitor, a MBOAT2 inhibitor, GPX465ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 2025 inhibitor, or any combination thereof and wherein the second inhibitor comprises a PTN inhibitor, a mTOR inhibitor, a mTORCl inhibitor, a RPTOR inhibitor, a SREBP1 inhibitor, a SCD1 inhibitor, or any combination thereof.

73. The method of any one of claims 55-72, wherein the first inhibitor comprises a composition comprising an inhibitory RNA for a messenger RNA sequence encoding a NKX3.1 protein and wherein the second inhibitor comprises a composition comprising an inhibitory RNA for a messenger RNA sequence encoding a PTN protein.

74. The method of any one of claims 55-73, wherein the first inhibitor comprises a NKX3. 1 inhibitor and wherein the second inhibitor comprises a PTN inhibitor.

75. The method of any one of claims 55-74, wherein the pharmaceutical composition is administered before, after, or in combination with an anti-androgen treatment.

76. The method of claim 75, wherein the anti-androgen treatment comprises administration of enzalutamide, abiraterone, apalutamide, bicalutamide, darolutamide, flutamide, or nilutamide.

77. The method of claim 75, wherein the anti-androgen treatment comprises administration of one or more luteinizing hormone-releasing hormone (LHRH) agonists.

78. The method of any one of claims 55-77, wherein the pharmaceutical composition is administered before, after, or in combination with radiation therapy.

79. The method of any one of claims 75-78, wherein the administration of the pharmaceutical composition to the subject in combination with an anti-androgen treatment decreases tumor size.

80. The method of any one of claims 75-79, wherein the administration of the pharmaceutical composition to the subject in combination with an anti-androgen treatment decreases tumor number.66ACTIVEUS 211605386v.lAttorney Docket No.: 0019240.01348WO1 Date of Electronic Filing: September 18, 202581. The method of any one of claims 75-80, wherein the administration of the pharmaceutical composition to the subject in combination with an anti-androgen treatment prevents cancer metastasis.

82. The method of any one of claims 1-81, wherein the subject is human.67ACTIVEUS 211605386v.l

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