Patient-derived Prostate Cancer Research Articles

Direct transposition of native DNA for sensitive multimodal single-molecule sequencing

Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.

Patient-derived castration-resistant prostate cancer model revealed CTBP2 upregulation mediated by OCT1 and androgen receptor

BackgroundProstate cancer is dependent on androgen receptor (AR) signaling, and androgen deprivation therapy (ADT) has proven effective in targeting prostate cancer. However, castration-resistant prostate cancer (CRPC) eventually emerges. AR signaling inhibitors (ARSI) have been also used, but resistance to these agents develops due to genetic AR alterations and epigenetic dysregulation.MethodsIn this study, we investigated the role of OCT1, a member of the OCT family, in an AR-positive CRPC patient-derived xenograft established from a patient with resistance to ARSI and chemotherapy. We conducted a genome-wide analysis chromatin immunoprecipitation followed by sequencing and bioinformatic analyses using public database.ResultsGenome-wide analysis of OCT1 target genes in PDX 201.1 A revealed distinct OCT1 binding sites compared to treatment-naïve cells. Bioinformatic analyses revealed that OCT1-regulated genes were associated with cell migration and immune system regulation. In particular, C-terminal Binding Protein 2 (CTBP2), an OCT1/AR target gene, was correlated with poor prognosis and immunosuppressive effects in the tumor microenvironment. Metascape revealed that CTBP2 knockdown affects genes related to the immune response to bacteria. Furthermore, TISIDB analysis suggested the relationship between CTBP2 expression and immune cell infiltration in prostate cancer, suggesting that it may contribute to immune evasion in CRPC.ConclusionsOur findings shed light on the genome-wide network of OCT1 and AR in AR-positive CRPC and highlight the potential role of CTBP2 in immune response and tumor progression. Targeting CTBP2 may represent a promising therapeutic approach for aggressive AR-positive CRPC. Further validation will be required to explore novel therapeutic strategies for CRPC management.

Quantifying Y chromosome loss in primary and metastatic prostate cancer by chromosome painting.

Somatic Y chromosome loss in hematopoietic cells is associated with higher mortality in men. However, the status of the Y chromosome in cancer tissue is not fully known due to technical limitations, such as difficulties in labelling and sequencing DNA from the Y chromosome. We have developed a system to quantify Y chromosome gain or loss in patient-derived prostate cancer organoids. Using our system, we observed Y chromosome loss in 4 of the 13 (31%) patient-derived metastatic castration-resistant prostate cancer (mCRPC) organoids; interestingly, loss of Yq (long arm of the Y chromosome) was seen in 38% of patient-derived organoids. Additionally, potential associations were observed between mCRPC and Y chromosome nullisomy. The prevalence of Y chromosome loss was similar in primary and metastatic tissue, suggesting that Y chromosome loss is an early event in prostate cancer evolution and may not a result of drug resistance or organoid derivation. This study reports quantification of Y chromosome loss and gain in primary and metastatic prostate cancer tissue and lays the groundwork for further studies investigating the clinical relevance of Y chromosome loss or gain in mCRPC.

Preclinical Efficacy of a PSMA-Targeted Actinium-225 Conjugate (225Ac-Macropa-Pelgifatamab): A Targeted Alpha Therapy for Prostate Cancer.

Initially, prostate cancer responds to hormone therapy, but eventually resistance develops. Beta emitter-based prostate-specific membrane antigen (PSMA)-targeted radionuclide therapy is approved for the treatment of metastatic castration-resistant prostate cancer. Here we introduce a targeted alpha therapy (TAT) consisting of the PSMA antibody pelgifatamab covalently linked to a macropa chelator and labeled with actinium-225 and compare its efficacy and tolerability with other TATs. The in vitro characteristics and in vivo biodistribution, antitumor efficacy, and tolerability of 225Ac-macropa-pelgifatamab (225Ac-pelgi) and other TATs were investigated in cell line- and patient-derived prostate cancer xenograft models. The antitumor efficacy of 225Ac-pelgi was also investigated in combination with the androgen receptor inhibitor darolutamide. Actinium-225-labeling of 225Ac-pelgi was efficient already at room temperature. Potent in vitro cytotoxicity was seen in PSMA-expressing (LNCaP, MDA-PCa-2b, and C4-2) but not in PSMA-negative (PC-3 and DU-145) cell lines. High tumor accumulation was seen for both 225Ac-pelgi and 225Ac-DOTA-pelgi in the MDA-PCa-2b xenograft model. In the C4-2 xenograft model, 225Ac-pelgi showed enhanced antitumor efficacy with a T/Cvolume (treatment/control) ratio of 0.10 compared with 225Ac-DOTA-pelgi, 225Ac-DOTA-J591, and 227Th-HOPO-pelgifatamab (227Th-pelgi; all at 300 kBq/kg) with T/Cvolume ratios of 0.37, 0.39, and 0.33, respectively. 225Ac-pelgi was less myelosuppressive than 227Th-pelgi. 225Ac-pelgi showed dose-dependent treatment efficacy in the patient-derived KuCaP-1 model and strong combination potential with darolutamide in both cell line- (22Rv1) and patient-derived (ST1273) xenograft models. These results provide a strong rationale to investigate 225Ac-pelgi in patients with prostate cancer. A clinical phase I study has been initiated (NCT06052306).

Dual targeting of the androgen receptor and PI3K/AKT/mTOR pathways in prostate cancer models improves antitumor efficacy and promotes cell apoptosis.

Prostate cancer is a frequent malignancy in older men and has a very high 5-year survival rate if diagnosed early. The prognosis is much less promising if the tumor has already spread outside the prostate gland. Targeted treatments mainly aim at blocking androgen receptor (AR) signaling and initially show good efficacy. However, tumor progression due to AR-dependent and AR-independent mechanisms is often observed after some time, and novel treatment strategies are urgently needed. Dysregulation of the PI3K/AKT/mTOR pathway in advanced prostate cancer and its implication in treatment resistance has been reported. We compared the impact of PI3K/AKT/mTOR pathway inhibitors with different selectivity profiles on in vitro cell proliferation and on caspase 3/7 activation as a marker for apoptosis induction, and observed the strongest effects in the androgen-sensitive prostate cancer cell lines VCaP and LNCaP. Combination treatment with the AR inhibitor darolutamide led to enhanced apoptosis in these cell lines, the effects being most pronounced upon cotreatment with the pan-PI3K inhibitor copanlisib. A subsequent transcriptomic analysis performed in VCaP cells revealed that combining darolutamide with copanlisib impacted gene expression much more than individual treatment. A comprehensive reversal of the androgen response and the mTORC1 transcriptional programs as well as a marked induction of DNA damage was observed. Next, an in vivo efficacy study was performed using the androgen-sensitive patient-derived prostate cancer (PDX) model LuCaP 35 and a superior efficacy was observed after the combined treatment with copanlisib and darolutamide. Importantly, immunohistochemistry analysis of these treated tumors showed increased apoptosis, as revealed by elevated levels of cleaved caspase 3 and Bcl-2-binding component 3 (BBC3). In conclusion, these data demonstrate that concurrent blockade of the PI3K/AKT/mTOR and AR pathways has superior antitumor efficacy and induces apoptosis in androgen-sensitive prostate cancer cell lines and PDX models.

Interleukin-30 subverts prostate cancer-endothelium crosstalk by fostering angiogenesis and activating immunoregulatory and oncogenic signaling pathways

BackgroundCancer-endothelial interplay is crucial for tumor behavior, yet the molecular mechanisms involved are largely unknown. Interleukin(IL)-30, which is expressed as a membrane-anchored cytokine by human prostate cancer (PC) cells, promotes PC vascularization and progression, but the underlying mechanisms have yet to be fully explored.MethodsPC-endothelial cell (EC) interactions were investigated, after coculture, by flow cytometry, transcriptional profiling, western blot, and ELISA assays. Proteome profiler phospho-kinase array unveiled the molecular pathways involved. The role of tumor-derived IL30 on the endothelium's capacity to generate autocrine circuits and vascular budding was determined following IL30 overexpression, by gene transfection, or its deletion by CRISPR/Cas9 genome editing. Clinical value of the experimental findings was determined through immunopathological study of experimental and patient-derived PC samples, and bioinformatics of gene expression profiles from PC patients.ResultsContact with PC cells favors EC proliferation and production of angiogenic and angiocrine factors, which are boosted by PC expression of IL30, that feeds autocrine loops, mediated by IGF1, EDN1, ANG and CXCL10, and promotes vascular budding and inflammation, via phosphorylation of multiple signaling proteins, such as Src, Yes, STAT3, STAT6, RSK1/2, c-Jun, AKT and, primarily CREB, GSK-3α/β, HSP60 and p53. Deletion of the IL30 gene in PC cells inhibits endothelial expression of IGF1, EDN1, ANG and CXCL10 and substantially impairs tumor angiogenesis. In its interaction with IL30-overexpressing PC cells the endothelium boosts their expression of a wide range of immunity regulatory genes, including CCL28, CCL4, CCL5, CCR2, CCR7, CXCR4, IL10, IL13, IL17A, FASLG, IDO1, KITLG, TNFA, TNFSF10 and PDCD1, and cancer driver genes, including BCL2, CCND2, EGR3, IL6, VEGFA, KLK3, PTGS1, LGALS4, GNRH1 and SHBG. Immunopathological analyses of PC xenografts and in silico investigation of 1116 PC cases, from the Prostate Cancer Transcriptome Atlas, confirmed the correlation between the expression of IL30 and that of both pro-inflammatory genes, NOS2, TNFA, CXCR5 and IL12B, and cancer driver genes, LGALS4, GNRH1 and SHBG, which was validated in a cohort of 80 PC patients.ConclusionsIL30 regulates the crosstalk between PC and EC and reshapes their transcriptional profiles, triggering angiogenic, immunoregulatory and oncogenic gene expression programs. These findings highlight the angiostatic and oncostatic efficacy of targeting IL30 to fight PC.

TGF-β induces matrisome pathological alterations and EMT in patient-derived prostate cancer tumoroids

Extracellular matrix (ECM) tumorigenic alterations resulting in high matrix deposition and stiffening are hallmarks of adenocarcinomas and are collectively defined as desmoplasia. Here, we thoroughly analysed primary prostate cancer tissues obtained from numerous patients undergoing radical prostatectomy to highlight reproducible structural changes in the ECM leading to the loss of the glandular architecture. Starting from patient cells, we established prostate cancer tumoroids (PCTs) and demonstrated they require TGF-β signalling pathway activity to preserve phenotypical and structural similarities with the tissue of origin. By modulating TGF-β signalling pathway in PCTs, we unveiled its role in ECM accumulation and remodelling in prostate cancer. We also found that TGF-β-induced ECM remodelling is responsible for the initiation of prostate cell epithelial-to-mesenchymal transition (EMT) and the acquisition of a migratory, invasive phenotype. Our findings highlight the cooperative role of TGF-β signalling and ECM desmoplasia in prompting prostate cell EMT and promoting tumour progression and dissemination.

Introduction of Androgen Receptor Targeting shRNA Inhibits Tumor Growth in Patient-Derived Prostate Cancer Xenografts.

Patient-derived xenograft (PDX) models have been established as important preclinical cancer models, overcoming some of the limitations associated with the use of cancer cell lines. The utility of prostate cancer PDX models has been limited by an inability to genetically manipulate them in vivo and difficulties sustaining PDX-derived cancer cells in culture. Viable, short-term propagation of PDX models would allow in vitro transfection with traceable reporters or manipulation of gene expression relevant to different studies within the prostate cancer field. Here, we report an organoid culture system that supports the growth of prostate cancer PDX cells in vitro and permits genetic manipulation, substantially increasing the scope to use PDXs to study the pathobiology of prostate cancer and define potential therapeutic targets. We have established a short-term PDX-derived in vitro cell culture system which enables genetic manipulation of prostate cancer PDXs LuCaP35 and BM18. Genetically manipulated cells could be re-established as viable xenografts when re-implanted subcutaneously in immunocompromised mice and were able to be serially passaged. Tumor growth of the androgen-dependent LuCaP35 PDX was significantly inhibited following depletion of the androgen receptor (AR) in vivo. Taken together, this system provides a method to generate novel preclinical models to assess the impact of controlled genetic perturbations and allows for targeting specific genes of interest in the complex biological setting of solid tumors.

UP17 - Overcoming docetaxel resistance in advanced prostate cancer: Analysis of statin therapy from a cell-based model to a patient derived prostate cancer tissue micro array

UP17 - Overcoming docetaxel resistance in advanced prostate cancer: Analysis of statin therapy from a cell-based model to a patient derived prostate cancer tissue micro array

The Bivalent Bromodomain Inhibitor MT-1 Inhibits Prostate Cancer Growth.

Bromodomains (BD) are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation of several genes including protooncogene cellular myelocytomatosis (c-Myc). c-Myc is difficult to target directly by agents due to its disordered alpha helical protein structure and predominant nuclear localization. The epigenetic targeting of c-Myc by BD inhibitors is an attractive therapeutic strategy for prostate cancer (PC) associated with increased c-Myc upregulation with advancing disease. MT-1 is a bivalent BD inhibitor that is 100-fold more potent than the first-in-class BD inhibitor JQ1. MT-1 decreased cell viability and causes cell cycle arrest in G0/G1 phase in castration-sensitive and resistant PC cell lines in a dose-dependent fashion. The inhibition of c-Myc function by MT-1 was molecularly corroborated by the de-repression of Protein Kinase D1 (PrKD) and increased phosphorylation of PrKD substrate proteins: threonine 120, serine 11, and serine 216 amino acid residues in β-Catenin, snail, and cell division cycle 25c (CDC25c) proteins, respectively. The treatment of 3D cell cultures derived from three unique clinically annotated heavily pretreated patient-derived PC xenografts (PDX) mice models with increasing doses of MT-1 demonstrated the lowest IC50 in tumors with c-Myc amplification and clinically resistant to Docetaxel, Cabazitaxel, Abiraterone, and Enzalutamide. An intraperitoneal injection of either MT-1 or in combination with 3jc48-3, an inhibitor of obligate heterodimerization with MYC-associated protein X (MAX), in mice implanted with orthotopic PC PDX, decreased tumor growth. This is the first pre-clinical study demonstrating potential utility of MT-1 in the treatment of PC with c-Myc dysregulation.

Abstract B065: Combining the androgen receptor inhibitor darolutamide with PI3K/AKT/mTOR pathway inhibitors has superior efficacy in preclinical models of prostate cancer

Abstract The phosphoinositide 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is dysregulated in the majority of advanced prostate cancer patients, often linked to phosphate and tensin homolog (PTEN) inactivation. An increasing number of studies demonstrates the reciprocal crosstalk between the PI3K/AKT/mTOR and the androgen receptor (AR) pathways with the inhibition of one of them leading to the compensatory activation of the other one. We compared the effects of different PI3K/AKT/mTOR pathway inhibitors as single agents or in combination with the AR inhibitor darolutamide.In vitro impact of the compounds was evaluated in prostate cancer cell lines by proliferation and apoptosis assays, and by RNA-seq analysis. In vivo effects were determined in a patient-derived (PDX) prostate cancer model in an efficacy study followed by immunohistochemistry analysis. Analysis of VCaP cells treated with the androgen R1881 showed that beside androgen response, the PI3K/AKT/mTOR pathway was an essential hallmark characteristic of androgen action. Additional darolutamide treatment opposed androgen effects on both pathways. We therefore evaluated the impact of selective inhibitors of PI3K or AKT and saw strong in vitro antiproliferative effects in the prostate cancer cell lines tested. We then focused on the pan-PI3K inhibitor copanlisib and observed pronounced synergistic effects with darolutamide in the VCaP model. This was accompanied by induction of PARP cleavage and activation of caspase 3/7. A detailed transcriptomic analysis revealed that the combination of both inhibitors resulted in more pronounced transcript changes, compared to individual treatments. Principal component analysis furthermore showed the combination to be closer to the DMSO control along the axis that represents androgen regulation. We observed a pronounced downregulation of cell proliferation and cell division genes following combination treatment compared to individual treatments, a prominent example being the proliferation marker KI67. In vivo studies performed with the PDX LuCaP 35 model revealed a superior inhibitory effect of the combination treatment with darolutamide and copanlisib when compared to single agents. Immunohistochemistry analysis revealed a significant upregulation of a pro-apoptotic pathway in tumors treated with darolutamide and copanlisib which was not observed in single treatment arms, when compared to vehicle control. In summary, we found that different PI3K and AKT inhibitors impaired the in vitro proliferation of prostate cancer cell lines. Combining the AR inhibitor darolutamide with the pan-PI3K inhibitor copanlisib led to increased apoptosis and down-regulation of cell division and proliferation genes. Importantly, the improved tumor inhibition by the combination was also observed in vivo. These results warrant further analysis of the impact of combined AR and PI3K pathway inhibition in prostate cancer, especially when aberrations in the PI3K/AKT/mTOR pathway or PTEN loss are observed. Citation Format: Simon Heller, Tatsuo Sugawara, Ekaterina Nevedomskaya, Simon J. Baumgart, Holly Nguyen, Eva Corey, Annika Böhme, Oliver von Ahsen, Oliver Politz, Bernard Haendler. Combining the androgen receptor inhibitor darolutamide with PI3K/AKT/mTOR pathway inhibitors has superior efficacy in preclinical models of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B065.

A novel biguanide derivative, IM176, induces prostate cancer cell death by modulating the AMPK-mTOR and androgen receptor signaling pathways

Metformin and phenformin, biguanide derivatives that are widely used to treat type 2 diabetes mellitus, have recently been shown to exert potential anticancer effects in prostate cancer. This study compared the anti-prostate cancer effects of the novel biguanide derivative IM176 with those of metformin and phenformin. Prostate cancer cell lines and patient-derived castration-resistant prostate cancer (CRPC) cells were treated with IMI76, metformin, and phenformin. The effects of these agents on cell viability, annexin V-FITC apoptosis, mTOR inhibition, protein expression and phosphorylation, and gene expression were evaluated. IM176 dose-dependently reduced the viability of all prostate cancer cell lines tested, with IC 50 s (LNCaP: 18.5 μM; 22Rv1: 36.8 μM) lower than those of metformin and phenformin. IM176 activated AMP-activated protein kinase (AMPK), inhibiting mTOR and reducing the phosphorylation of p70S6K1 and S6. IM176 inhibited the expression of androgen receptor (AR), the AR splice variant 7 (AR-V7) and prostate-specific antigen in LNCaP and 22Rv1 cells. IM176 increased caspase-3 cleavage and annexin V-positive/PI-positive cells, which indicated apoptosis. Moreover, IM176 reduced viability, with low IC 50 , in cultured cells derived from two patients with CRPC. The antitumor effects of IM176 were comparable with those of other biguanides. IM176 may therefore be a novel candidate for the treatment of patients with prostate cancer, including those with CRPC.

Integrative multi-omics and drug–response characterization of patient-derived prostate cancer primary cells

Prostate cancer (PCa) is the second most prevalent malignancy in males across the world. A greater knowledge of the relationship between protein abundance and drug responses would benefit precision treatment for PCa. Herein, we establish 35 Chinese PCa primary cell models to capture specific characteristics among PCa patients, including gene mutations, mRNA/protein/surface protein distributions, and pharmaceutical responses. The multi-omics analyses identify Anterior Gradient 2 (AGR2) as a pre-operative prognostic biomarker in PCa. Through the drug library screening, we describe crizotinib as a selective compound for malignant PCa primary cells. We further perform the pharmacoproteome analysis and identify 14,372 significant protein-drug correlations. Surprisingly, the diminished AGR2 enhances the inhibition activity of crizotinib via ALK/c-MET-AKT axis activation which is validated by PC3 and xenograft model. Our integrated multi-omics approach yields a comprehensive understanding of PCa biomarkers and pharmacological responses, allowing for more precise diagnosis and therapies.

Abstract 4332: Characterizing cancer-associated fibroblasts in prostate cancer using Vizgen’s MERSCOPETM Platform

Abstract Understanding the spatial complexities within cancer provides crucial information for evaluating individual tumor development and progression; recent research in prostate cancer has shown how critical it is for oncologists to maintain a spatial context while characterizing a highly heterogeneous tumor microenvironment. As more and more research groups acknowledge the need for spatial biology solutions to solve difficult questions surrounding cell identity, heterogeneity, and interaction, Vizgen’s MERSCOPETM Platform provides a powerful tool to illuminate spatial information. Built on Multiplexed Error-Robust Fluorescence in situ Hybridization (MERFISH) technology, Vizgen’s MERSCOPETM enables the direct profiling of the spatial organization of intact tissue with subcellular resolution. Here, we use a 500-gene panel to demonstrate the MERSCOPE’s spatial capability, assessing the canonical signaling pathways of cancer, cancer type-specific genes, select immune genes, proto-oncogenes, and tumor-suppressor genes in patient-derived prostate cancer samples. Using the Vizgen Cell Boundary Stain kit, we were able to generate a molecular and cellular atlas of these individual patient tumors by clustering cells based on gene expression and mapping these clusters back onto a spatial representation of the tumor. We found specific clusters relating to prognostic outcomes in cancer-associated fibroblasts (CAFs) and myofibroblasts. Subsequently, we used single-cell RNA-seq datasets to impute related transcriptome data for these various fibroblast clusters to help characterize them more specifically. To understand the heterogeneity intrinsic to CAFs, we performed a neighborhood analysis to determine how proximity to immune cells, tumor cells, and other stromal cells altered gene expression in different CAF clusters. We found gene expression changes associated with inflammation and reactive stromal signaling in CAFs based on their proximity to tumor versus stromal tissue compartments. We also compared the gene expression of tumor cells near CAFs to those more deeply embedded within the tumor compartment. This revealed interesting transcriptional covariations between superficial layers of tumor and their surrounding stromal milieu involved in cell proliferation and signaling. These analyses demonstrate the transcriptional variation possible across fibroblastic stromal cells acting in tumor-adjacent or stromal contexts. Notably, we were able to map and catalogue different patterns of gene expression relating to inflammation, tumor proliferation, and reactive stromal signaling. These findings demonstrate how the MERSCOPE platform can provide deep insights into the complex heterogeneity observed in the tumor microenvironment. Citation Format: Ben Patterson, Cheng-Yi Chen, Nicolas Fernandez, Simran Kaushal, Leiam Colbert, Jiang He. Characterizing cancer-associated fibroblasts in prostate cancer using Vizgen’s MERSCOPETM Platform. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4332.

PD04-09 COMBINATION TREATMENT WITH HSP70/STUB1 MODULATOR AND ENZALUTAMIDE YIELDS SYNERGISTIC ACTIVITY IN ADVANCED PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023PD04-09 COMBINATION TREATMENT WITH HSP70/STUB1 MODULATOR AND ENZALUTAMIDE YIELDS SYNERGISTIC ACTIVITY IN ADVANCED PROSTATE CANCER Pengfei Xu, Joy C. Yang, Bo Chen, Christopher Nip, Shu Ning, Allen C. Gao, Hao Shao, Jason Gestwicki, Christopher P. Evans, and Chengfei Liu Pengfei XuPengfei Xu More articles by this author , Joy C. YangJoy C. Yang More articles by this author , Bo ChenBo Chen More articles by this author , Christopher NipChristopher Nip More articles by this author , Shu NingShu Ning More articles by this author , Allen C. GaoAllen C. Gao More articles by this author , Hao ShaoHao Shao More articles by this author , Jason GestwickiJason Gestwicki More articles by this author , Christopher P. EvansChristopher P. Evans More articles by this author , and Chengfei LiuChengfei Liu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Ubiquitin proteasome system is suppressed in enzalutamide resistant prostate cancer cells and that the HSP70/STUB1 machinery is involved in androgen receptor (AR) and AR variant protein stabilization. Targeting HSP70 could be a valuable strategy to overcome the resistance of androgen receptor signaling inhibitors (ARSI) in advanced prostate cancer. In this study, we tested novel HSP70/STUB1modulators in enzalutamide resistant cells and patient-derived prostate cancer cell models. METHODS: Expression of AR-V7, HSP70 and STUB1 were determined by qRT-PCR and western blot. Expression of HSP70 and STUB1 was down-regulated using specific siRNA. HSP70/STUB1 and AR-V7 interaction was determined by co-immunoprecipitation and dual immunofluorescence. Tumor organoids were established from the patient derived xenografts (PDX) and the viability was stained by LIVE/DEAD™ Cell Imaging Kit. The effects of HSP70 inhibitors on enzalutamide sensitivity were examined in cell line model and patient-derived cell models. RESULTS: A novel HSP70 inhibitor JG98 significantly suppressed drug resistant C4-2B MDVR and CWR22Rv1 cell growth and enhanced enzalutamide treatment (p<0.001). JG98 also suppressed the PDX derived organoid growth and induced organoid death in a dose dependent manner (p<0.001). Mechanistically, JG98 suppressed AR/AR-V7 expression in resistant cells and promoted STUB1 entering into the nucleus and bound to AR-V7. Knockdown of STUB1 diminished the anti-cancer effect and AR-V7 inhibition by JG98. A more potent HSP70 inhibitor JG231 was developed from JG98. JG231 significantly improved the drug solubility and showed better pharmacokinetic characteristics than JG98. Moreover, JG231 effectively suppressed the cell growth and enhanced enzalutamide treatment in resistant cells and PDX derived prostate cancer cells (p<0.001). CONCLUSIONS: The HSP70/STUB1 machinery involved in AR/AR-V7 regulation and enzalutamide resistance. Targeting HSP70 by novel HSP70/STUB1 modulators overcome the ARSI resistance and improve their therapy. Source of Funding: This work was supported in part by grants NIH/NCI R37CA249108 (C, Liu) and R01CA251253 (C, Liu) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e145 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Pengfei Xu More articles by this author Joy C. Yang More articles by this author Bo Chen More articles by this author Christopher Nip More articles by this author Shu Ning More articles by this author Allen C. Gao More articles by this author Hao Shao More articles by this author Jason Gestwicki More articles by this author Christopher P. Evans More articles by this author Chengfei Liu More articles by this author Expand All Advertisement PDF downloadLoading ...

Metabolite-Specific Echo Planar Imaging for Preclinical Studies with Hyperpolarized 13C-Pyruvate MRI.

Metabolite-specific echo-planar imaging (EPI) sequences with spectral-spatial (spsp) excitation are commonly used in clinical hyperpolarized [1-13C]pyruvate studies because of their speed, efficiency, and flexibility. In contrast, preclinical systems typically rely on slower spectroscopic methods, such as chemical shift imaging (CSI). In this study, a 2D spspEPI sequence was developed for use on a preclinical 3T Bruker system and tested on in vivo mice experiments with patient-derived xenograft renal cell carcinoma (RCC) or prostate cancer tissues implanted in the kidney or liver. Compared to spspEPI sequences, CSI were found to have a broader point spread function via simulations and exhibited signal bleeding between vasculature and tumors in vivo. Parameters for the spspEPI sequence were optimized using simulations and verified with in vivo data. The expected lactate SNR and pharmacokinetic modeling accuracy increased with lower pyruvate flip angles (less than 15°), intermediate lactate flip angles (25° to 40°), and temporal resolution of 3 s. Overall SNR was also higher with coarser spatial resolution (4 mm isotropic vs. 2 mm isotropic). Pharmacokinetic modelling used to fit kPL maps showed results consistent with the previous literature and across different sequences and tumor xenografts. This work describes and justifies the pulse design and parameter choices for preclinical spspEPI hyperpolarized 13C-pyruvate studies and shows superior image quality to CSI.

Treatment of Prostate Cancer with CD46-targeted 225Ac Alpha Particle Radioimmunotherapy.

Radiopharmaceutical therapy is changing the standard of care in prostate cancer and other malignancies. We previously reported high CD46 expression in prostate cancer and developed an antibody-drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody. [225Ac]DOTA-YS5 was developed, and its therapeutic efficiency was tested on cell-derived (22Rv1, DU145), and patient-derived (LTL-545, LTL484) prostate cancer xenograft models. Biodistribution studies were carried out on 22Rv1 tumor xenograft models to confirm the targeting efficacy. Toxicity analysis of the [225Ac]DOTA-YS5 was carried out on nu/nu mice to study short-term (acute) and long-term (chronic) toxicity. Biodistribution study shows that [225Ac]DOTA-YS5 agent delivers high levels of radiation to the tumor tissue (11.64% ± 1.37%ID/g, 28.58% ± 10.88%ID/g, 29.35% ± 7.76%ID/g, and 31.78% ± 5.89%ID/g at 24, 96, 168, and 408 hours, respectively), compared with the healthy organs. [225Ac]DOTA-YS5 suppressed tumor size and prolonged survival in cell line-derived and patient-derived xenograft models. Toxicity analysis revealed that the 0.5 μCi activity levels showed toxicity to the kidneys, likely due to redistribution of daughter isotope 213Bi. [225Ac]DOTA-YS5 suppressed the growth of cell-derived and patient-derived xenografts, including prostate-specific membrane antigen-positive and prostate-specific membrane antigen-deficient models. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46-targeted radioligand therapy in prostate cancer.

181 (PB061) - Spatial transcriptomics analysis of co-targeted PIM and PI3 K/mTOR in multikinase inhibitor and single kinase inhibitor combination-treated patient-derived prostate cancer explants

Prostate cancer is a multifocal heterogeneous disease that remains the second leading cause of male death worldwide. These deaths are caused mainly by metastasis. Moreover, PIM and PI3 K/mTOR pathways are frequently dysregulated in prostate cancer, and may lead to an invasion, increased metastasis and decreased survival. However, the interconnection of these pathways has been shown to mediate anti-tumour drug resistance. Furthermore, current treatments exhibit issues with toxicity. The novel preclinical multikinase PIM/PI3 K/mTOR inhibitor, AUM302, versus clinical trial investigated - a combination of the PI3 K/mTOR inhibitor BEZ235 (Dactolisib) and PIM inhibitor, AZD-1208 has been analyzed in our laboratory using a cohort of cancer explants emanating from our PEOPLE: PatiEnt prOstate samPLes for rEsea ch study and our current SCREEN study.

99P The transcriptional atlas of co-targeted PIM/PI3K/mTOR ex-vivo patient-derived prostate cancer as revealed by spatial transcriptomics

PIM 1 and PI3K/mTOR pathways are frequently dysregulated in prostate cancer and may lead to decreased survival invasion and metastasis. Moreover, anti-tumour drug resistance has been associated with the interconnection of these pathways. Furthermore, current treatments exhibit issues with toxicity. Hence, these pathways were co-targeted with novel preclinical multikinase PIM/PI3K/mTOR inhibitor- AUM302, PI3K/mTOR inhibitor BEZ235 (Dactolisib) and PIM inhibitor, AZD-1208 in our laboratory using a cohort of cancer explants emanating from our PEOPLE: PatiEnt prOstate samPLes for rEsea ch study and our current SCREEN study.

Abstract 1625: c-Myc inhibition in prostate cancer cell lines

Abstract The proto-oncogene c-Myc is a transcription factor that is upregulated in several human cancers. Despite its key role, therapeutic targeting of c-Myc remains a challenge because of a disordered protein tertiary structure. The basic helical structure and zipper protein of c-Myc forms an obligate heterodimer with its partner MAX to function as a transcription factor. An attractive strategy is to inhibit MYC/MAX dimerization to decrease c-Myc transcriptional function. Several methods have been described to inhibit MYC/MAX dimerization including small molecular inhibitors and proteomimetics. We used NOD SCID gamma mice (NGS mice) that are immunodeficient, non-obese diabetic laboratory mice (Jackson Laboratories, Cat Number J000077451) carrying patient-derived xenograft (PDX) following implantation of grade IV adenocarcinoma of the prostate from a patient into the flanks of each mouse. For maximum tolerable dose studies, we used C57BL/6 wild type mice (Jackson Laboratories, Cat Number J000664). We performed a maximum dose tolerability study with the drug 3JC48-3 up to 100 mg/kg to determine limiting toxicity. We monitored the rate of growth for 2 weeks and measured the final volume of the tumors. Immunoprecipitation studies were performed using the prostate cancer cell line, PC-3, that were treated with the drug 3JC48-3 at different doses (0, 5, 10, and 20uM) for 24 hours and subsequently lysed. We found 3JC48-3 decreases prostate cancer growth and viability in a dose-dependent fashion in vitro. We confirmed the inhibition of MYC/MAX dimerization by 3JC48-3 using immunoprecipitation experiments. Normal mice and mice with patient-derived prostate cancer xenografts (PDX) tolerated intraperitoneal injections of 3JC48-3 up to 100 mg/kg body weight without dose-limiting toxicity. Preliminary results in these PDX mouse models suggest that 3JC48-3 may be effective in decreasing the rate of tumor growth. Our study demonstrates that 3JC48-3 is a potent MYC/MAX heterodimerization inhibitor that decreases prostate cancer growth and viability in association with upregulation of both PrKD1 expression and kinase activity. In addition, 3JC48-3 is well tolerated in prostate cancer PDX mice models and may be a candidate drug for further preclinical development. Citation Format: Carlos Riveros, Victor Chalfant, Sanjeev Shukla, Teruko Osumi, KC Balaji. c-Myc inhibition in prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1625.