(A) Total dehydrated prostate weights from 6- and 10-week-old mice were averaged for each genotype (n = 3 to 6 mice per arm, mean ± SEM). wild-type, WT. (B) Phenotypical penetrance percentages for low-grade prostatic intraepithelial neoplasia (LgPIN), HgPIN, and cancer in anterior prostate tissues from 6- and 10-week-old mice evaluated by hematoxylin and eosin (H&E) staining. (C) Left: Representative ultrasound images of prostate tumors at 7 months outlined in yellow from indicated genotypes; scale bars, 9 mm. Right: Quantification of prostate tumor size in mice with an average age of 8 months (n = 5 mice per arm, mean ± SEM). (D) Kaplan-Meier survival curves for mice with the indicated genotypes. Dotted line highlights the median life span of 75 weeks for Pten^fl/fl;Myc^Tg mice. (E) Top: Representative bright-field images of three-dimensional organoid structures 6 days after seeding; scale bars, 50 μm. Bottom: Western blot analyzing the organoids, showing P-AKT, PTEN, GFP for Myc^Tg, and β-actin. (F) Newly synthesized proteins measured by ³⁵S methionine/cysteine incorporation in organoids (left panel), with quantification relative to WT littermates (right panel) (n = 5, mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, t test.

(A) Left: Representative IF images of P-PERK/cytokeratin 5 (CK5) or P-eIF2α/CK5 co-staining with DAPI (4′,6-diamidino-2-phenylindole) used to visualize the nuclei within anterior prostate tissue from 10-week-old mice; scale bars, 100 μm. P-PERK or P-eIF2α expression quantified relative to DAPI (n = 3 mice per arm, with four images averaged per mouse, mean ± SEM). (B) Representative IF images of P-eIF2α/CK5 co-staining with DAPI in anterior prostate tissue from 6-week-old mice (scale bars, 100 μm) (left panel) and directly within areas of PIN (right panel). Lower panel depicts a model showing the timeline of PCa development within Pten^fl/fl;Myc^Tg mice, highlighting when P-eIF2α is expressed. **P < 0.01, t test.

(A) Representative Western blot highlighting PERK signaling in organoid cultures. (B) Left: Total newly synthesized proteins measured by ³⁵S methionine/cysteine incorporation and Western blot showing P-eIF2α and ATF4 in organoids treated with dimethyl sulfoxide (DMSO) or ISRIB (500 nM) for 6 hours. Right: Quantification of radioactive pulse relative to loading, depicted as percent over WT (n = 3, mean ± SEM). (C) Schematic of preclinical trial for escalating dosage and MRI schedule over 6 weeks. (D) Representative scans of two ISRIB-treated mice after 1 and 6 weeks of treatment for comparison. Tumor is outlined in red, and arrows highlight the seminal vesicles (SV) surrounding the tumor. (E) Quantification of tumor size as fold change relative to baseline volume at 3- and 6-week time points (mean ± SEM). **P < 0.01, ***P < 0.001, t test.

(A) Representative Western blot showing PTEN, MYC, P-PERK, P-eIF2α, and total eIF2α expression with β-actin loading control (Ctrl) in human prostatic RWPE-1 cell lines. (B) Quantification of annexin V–positive cells analyzed by flow cytometry relative to control cells after treatment with DMSO or 500 nM ISRIB for 9 hours (n = 3, mean ± SEM) *P < 0.05, t test. (C) Kaplan-Meier analysis of clinical progression–free survival [progression defined as visceral or bone metastasis or PCSM] for patients with normal PTEN expression versus PTEN loss and relative MYC expression identified by IF from the TMA. (D) Kaplan-Meier analysis of MET/PCSM for patients with normal PTEN expression versus PTEN loss grouped by eIF2α phosphorylation. (E) Cox proportional hazards regression results are shown in a Forest plot of hazard ratios and 95% CI for factors associated with risk of clinical progression after surgery. Independent factors are tumor with PTEN loss/low P-eIF2α or PTEN loss/high P-eIF2α versus a reference group with normal PTEN expression; age in years; PSA in nanograms per milliliter; Gleason score > 7 versus 6; and pathological stage T3-T4 versus T2 at the time of prostatectomy.

(A) Schematic highlighting origin of PDX tumors from primary (pPCa) or lymph node metastasis (mPCa). Tumors from selected patients with high-risk features, based on Gleason score and clinical stage or with lymph node metastases determined by ⁶⁸Ga–PSMA-11 positron emission tomography (PET) scans, were used to generate PDXs. Primary and metastatic tumors were confirmed from tissue collected at the time of surgery and immediately implanted into immunodeficient NOD scid gamma (NSG) mice. (B) Representative IF images of MYC/CK8 (epithelial cell marker), P-AKT/CK8, or P-eIF2α/CK8, co-staining with DAPI from benign (Ben) tissue adjacent to pPCa or mPCa tumors; scale bars, 50 μm. Right: Quantification of protein expression as relative mean IF intensity normalized to adjacent stromal tissue. (C) Kaplan-Meier tumor survival curves for mice bearing pPCa or mPCa tumors treated with ISRIB (10 mg/kg) or vehicle (n = 8, per cohort; **P = 0.01, log-rank test). The survival curves represent mice euthanized when tumors reached an end point of 2 cm or when the mice showed clear signs of morbidity. (D) Representative tumor sizes after 10 days of treatment. (E) Representative TUNEL staining and quantification of PDX tumors treated with vehicle or ISRIB (10 mg/kg); scale bars, 100 μm (n = 3, ***P < 0.001, t test). (F) Quantification of newly synthesized proteins in vivo, assessed by incorporation of OP-Puro within PDX treated with ISRIB (10 mg/kg) or vehicle (n = 3 to 4 per arm, mean ± SEM; *P < 0.05, t test). n.s., not significant. MFI, mean fluorescence intensity.

(A) Representative H&E staining and IF of P-eIF2α, PTEN, and MYC expression at the primary site of implantation (mCRPC tumor), left kidney, and distant metastatic lesions in the liver; scale bars, 200 μm (top left); 100 μm (bottom left); and 50 μm for IF images. (B) Schematic of preclinical trial for mCRPC tumor growth and PET/CT schedule. Representative ⁶⁸Ga–PSMA-11 PET/CT scans on day 0 (time of treatment) and on day 7 are shown for the control versus ISRIB-treated cohorts. Uptake of the PSMA-targeted radiotracer agent is observed in the liver, lymph node, and at the site of primary tumor implantation in the left kidney capsule. Physiologic uptake of the PSMA-targeted radiotracer is also seen in the contralateral kidney and bladder because it is excreted in the urinary tract. Histologic confirmation of liver metastasis is shown by H&E staining at the time of euthanasia with dashed outlines around metastatic lesions. (C) Kaplan-Meier survival curve for mice bearing mCRPC tumors treated once per day with ISRIB (10 mg/kg) or vehicle (n = 3, per cohort); *P = 0.02, log-rank test. The survival curves represent mice euthanized when PSMA ⁶⁸Ga PET/CT showed progression from one distant metastatic lesion to two or more sites or when the mice showed signs of becoming moribund. (D) Quantification of visible metastatic lesions on the left medial lobe of the liver at the time of euthanasia in the cohorts (n = 3 per cohort); ***P = 0.001.