Prostate Cancer Patient-derived Xenografts 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.

DB-1310, an ADC comprised of a novel anti-HER3 antibody conjugated to a DNA topoisomerase I inhibitor, is highly effective for the treatment of HER3-positive solid tumors

BackgroundHER3 (ErbB3), a member of the human epidermal growth factor receptor family, is frequently overexpressed in various cancers. Multiple HER3-targeting antibodies and antibody–drug conjugates (ADCs) were developed for the solid tumor treatment, however none of HER3-targeting agent has been approved for tumor therapy yet. We developed DB-1310, a HER3 ADC composed of a novel humanized anti-HER3 monoclonal antibody covalently linked to a proprietary DNA topoisomerase I inhibitor payload (P1021), and evaluate the efficacy and safety of DB-1310 in preclinical models.MethodsThe binding of DB-1310 to Her3 and other HER families were measured by ELISA and SPR. The competition of binding epitope for DB-1310 and patritumab was tested by FACS. The sensitivity of breast, lung, prostate and colon cancer cell lines to DB-1310 was evaluated by in vitro cell killing assay. In vivo growth inhibition study evaluated the sensitivity of DB-1310 to Her3 + breast, lung, colon and prostate cancer xenograft models. The safety profile was also measured in cynomolgus monkey.ResultsDB-1310 binds HER3 via a novel epitope with high affinity and internalization capacity. In vitro, DB-1310 exhibited cytotoxicity in numerous HER3 + breast, lung, prostate and colon cancer cell lines. In vivo studies in HER3 + HCC1569 breast cancer, NCI-H441 lung cancer and Colo205 colon cancer xenograft models showed DB-1310 to have dose-dependent tumoricidal activity. Tumor suppression was also observed in HER3 + non-small cell lung cancer (NSCLC) and prostate cancer patient-derived xenograft (PDX) models. Moreover, DB-1310 showed stronger tumor growth-inhibitory activity than patritumab deruxtecan (HER3-DXd), which is another HER3 ADC in clinical development at the same dose. The tumor-suppressive activity of DB-1310 synergized with that of EGFR tyrosine kinase inhibitor, osimertinib, and exerted efficacy also in osimertinib-resistant PDX model. The preclinical assessment of safety in cynomolgus monkeys further revealed DB-1310 to have a good safety profile with a highest non severely toxic dose (HNSTD) of 45 mg/kg.ConclusionsThese finding demonstrated that DB-1310 exerted potent antitumor activities against HER3 + tumors in in vitro and in vivo models, and showed acceptable safety profiles in nonclinical species. Therefore, DB-1310 may be effective for the clinical treatment of HER3 + solid tumors.

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).

Abstract 4157: Combining hyperpolarized magnetic resonance and positron emission tomography to interrogate prostate cancer metabolism

Abstract Background: There is an unmet clinical need for imaging biomarkers to distinguish indolent from aggressive prostate cancer (PCa). Many advanced and aggressive PCa patients receiving anti-androgens (enzalutamide) develop resistance. It is hypothesized that dysregulated cell metabolism is a key driver for PCa progression and therapy resistance. Two pathways commonly involved in PCa are glycolysis and fatty acid metabolism. Therefore, metabolic imaging and metabolomics were performed on enzalutamide sensitive/resistant, Androgen Receptor dependent (AR+) and AR independent (AR-) patient derived xenograft (PDX) tumors. To interrogate both pathways, [1-13C]-pyruvate hyperpolarized magnetic resonance spectroscopy (HP-MRS) and [18F]-fluorodeoxyglucose positron emission tomography (18F-FDG) for glycolysis, and [18F]-fluoro-pivalic acid positron emission tomography (18F-FPIA) for fatty acid metabolism were employed. 1H Nuclear Magnetic Resonance (NMR) spectroscopy and liquid chromatography with tandem mass spectrometry (LC-MS-MS) were employed for validation purposes. Methods: [1-13C]-labeled pyruvic acid was hyperpolarized using a DNP HyperSense polarizer following standard protocol. Anatomical MRI and 13C-MRS were obtained on different PCa PDX mouse models at two different time points using a Bruker 7T scanner. The PDX models included AR+ enzalutamide sensitive (183-A, 180-30), AR+ enzalutamide resistant (274-4), and AR- (144-4, 114-B). The time points imaged were before and after 7 days of treatment with enzalutamide. 1H-NMR spectroscopy was performed on extracted tissue. Simultaneously, 18F-FPIA-PET imaging were acquired on the same models on an Albira trimodal PET station. Results: The dynamic metabolic flux ratio, lactate-to-pyruvate (Lac/Pyr) was determined in vivo and used as a treatment response marker. The Lac/Pyr ratios were significantly higher in resistant tumors compared to sensitive tumors (p<0.01). The enzalutamide sensitive group had a lower Lac/Pyr ratio after treatment, while the enzalutamide resistant group had a higher Lac/Pyr ratio. After treatment, there was also a decrease in [18F]-FDG uptake, corresponding to the HP-MRS data. This was expected as both Pyruvate and [18F]-FDG interrogate the glycolytic pathway. As for the fatty acid metabolic imaging, PET imaging also revealed that [18F]-FPIA is transported into the tumors, and we are currently exploring how its uptake varies between AR (+/-) PCa-PDX tumors and enzalutamide resistant/sensitive models. Ex vivo NMR and mass spectrometry-based metabolomics validated the in vivo HP-MRS data with higher lactate levels in drug resistant tumors. Conclusion: Combining HP-MRS and PET presents an exciting opportunity to realize imaging-based personalized medicine in different AR (+/-) PCa-PDX preclinical models by interrogating glycolysis and fatty acid oxidation pathways. Citation Format: Muxin Wang, José S. Enriquez, Prasanta Dutta, Jenny Han, Peter Shepherd, Daniel Frigo, Federica Pisaneschi, Pratip K. Bhattacharya. Combining hyperpolarized magnetic resonance and positron emission tomography to interrogate prostate cancer metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4157.

Abstract 5968: CDC7 is a targetable regulator of advanced prostate cancer

Abstract Prostate cancer is estimated to contribute to over 34,000 deaths in men residing in the United States, with the majority fatality due to metastatic disease. CDC7 is a kinase that regulates DNA replication and is an emerging cancer biomarker for poor prognosis in carcinomas such as Wilms tumor and hepatocellular carcinomas. In this study, we demonstrated that high level of CDC7 is associated with metastatic prostate cancer when compared to benign prostate tissues or localized prostate cancer. Furthermore, we found that CDC7 expression is highest in prostate cancer patient-derived xenografts (PDX) models of adenocarcinomas with neuroendocrine features. In vitro, we showed that inhibition of CDC7 with TAK-931, a CDC7 specific inhibitor, reduced the ability of aggressive prostate cancer cells to proliferate, migrate, and invade. Similarly, knock-down of CDC7 in prostate cancer cell lines inhibited cell growth in a colony formation assay. TAK-931 treated prostate cancer cell lines also showed an abnormal cell cycle profile, indicating that inhibiting CDC7 in aggressive prostate cancer could contribute to replication stress and promote apoptosis. Overall, this study demonstrates that CDC7 is a targetable protein that regulates advanced and aggressive prostate cancer growth. Citation Format: Alifiani Bonita Hartono, Sidharth Paparaju, Chung Lee, Shiqin Liu, Busola R. Alabi, En-Chi Hsu, James D. Brooks, Eva Corey, Tanya Stoyanova. CDC7 is a targetable regulator of advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5968.

Abstract 6913: A preclinical platform of prostate cancer PDX and derived cellular models as a tool for pharmacological screening and functional studies

Abstract Prostate Cancer (PCa) is the most frequently diagnosed cancer in men and remains one of the leading causes of cancer death worldwide. Despite undeniable progress in understanding the biology and genetics of this highly heterogenous and complex disease, the development of effective therapies is hampered by the lack of relevant experimental models recapitulating its diversity. To tackle this issue, over the last years, we have generated and characterized at the molecular and pharmacology level a collection of prostate cancer patient-derived xenografts (PDX) that accurately reproduce PCa histological and molecular heterogeneity. Although the development of these PCa PDX has been challenging, we are now able to offer a preclinical platform of 11 fully characterized PCa PDX models and in vitro cellular counterparts for preclinical evaluation of novel treatment modalities. Our platform consists of a collection of 4 androgen receptor high (ARPC), 2 androgen receptor low (ARLPC), 1 amphicrine prostate carcinoma (AMPC) and 4 castration-resistant neuroendocrine prostate cancer (CRPC-NE) PDX models and 6 cellular models derived from these PDX (PDXDC). Most of these PDX models were obtained by transplantation of post-surgery tumor specimens either by grafting of tumor fragments in the interscapular region of nude mice or in the subrenal capsule of NOD scid gamma mice. One model is a CTC-derived eXplant established using circulating tumor cells (CTCs) obtained by diagnostic leukapheresis injected into the fat pad of NOD scid gamma mice. PDXDC were obtained from dissociated PDX tumors cultured under various media and matrix conditions. They were characterized by comparison with the parental PDX by Short Tandem Repeat (STR) profiling before performing a master bank. All models (PDX and PDXDC) were characterized at molecular level by whole exome and RNA sequencing. PDX were characterized pharmacologically for response to standards of care, physical castration and response to the androgen receptor inhibitor enzalutamide. In vitro drug sensitivity of PDXDC was compared with their parental PDX in vivo drug response. This unique PDX panel reflects the molecular heterogeneity of prostate cancer and reproduces the molecular and drug response profile of human tumors. Associated to in vitro cell derivatives, our collection provides a powerful preclinical platform for translational research to identify mechanisms underlying acquired resistance to current therapies and develop novel treatment strategies against PCa and CRPC. Citation Format: Emilie Indersie, Léa Sinayen, Ludovic Bigot, Katell Mevel, Aurore Gorse, Marie Tavernier, Enora Le Ven, Luc Friboulet, Françoise Farace, Ester Morgado, Laurent Pouyet, Yohann Loriot, François Romagné, Olivier Déas. A preclinical platform of prostate cancer PDX and derived cellular models as a tool for pharmacological screening and functional studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6913.

Integrative Molecular Analyses of the MD Anderson Prostate Cancer Patient-derived Xenograft (MDA PCa PDX) Series.

Develop and deploy a robust discovery platform that encompasses heterogeneity, clinical annotation, and molecular characterization and overcomes the limited availability of prostate cancer models. This initiative builds on the rich MD Anderson (MDA) prostate cancer (PCa) patient-derived xenograft (PDX) resource to complement existing publicly available databases by addressing gaps in clinically annotated models reflecting the heterogeneity of potentially lethal and lethal prostate cancer. We performed whole-genome, targeted, and RNA sequencing in representative samples of the same tumor from 44 PDXs derived from 38 patients linked to donor tumor metadata and corresponding organoids. The cohort includes models derived from different morphologic groups, disease states, and involved organ sites (including circulating tumor cells), as well as paired samples representing heterogeneity or stages before and after therapy. The cohort recapitulates clinically reported alterations in prostate cancer genes, providing a data resource for clinical and molecular interrogation of suitable experimental models. Paired samples displayed conserved molecular alteration profiles, suggesting the relevance of other regulatory mechanisms (e.g., epigenomic) influenced by the microenvironment and/or treatment. Transcriptomically, models were grouped on the basis of morphologic classification. DNA damage response-associated mechanisms emerged as differentially regulated between adenocarcinoma and neuroendocrine prostate cancer in a cross-interrogation of PDX/patient datasets. We addressed the gap in clinically relevant prostate cancer models through comprehensive molecular characterization of MDA PCa PDXs, providing a discovery platform that integrates with patient data and benchmarked to therapeutically relevant consensus clinical groupings. This unique resource supports robust hypothesis generation and testing from basic, translational, and clinical perspectives.

Unraveling the Global Proteome and Phosphoproteome of Prostate Cancer Patient-Derived Xenografts.

Overall, our study highlights the importance of protein-based identification when compared with RNA and provides a rich resource of new and feasible targets for clinical assay development and in understanding the underlying biology of these tumors.

Patient-Derived Xenograft Models for Translational Prostate Cancer Research and Drug Development.

Patient-derived xenografts (PDXs) are a valuable preclinical research platform generated through transplantation of a patient's resected tumor into an immunodeficient or humanized mouse. PDXs serve as a high-fidelity avatar for both precision medicine and therapeutic testing against the cancer patient's disease state. While PDXs show mixed response to initial establishment, those that successfully engraft and can be sustained with serial passaging form a useful tool for basic and translational prostate cancer (PCa) research. While genetically engineered mouse (GEM) models and human cancer cell lines, and their xenografts, each play beneficial roles in discovery science and initial drug screening, PDX tumors are emerging as the gold standard approach for therapeutic proof-of-concept prior to entering clinical trial. PDXs are a powerful platform, with PCa PDXs shown to represent the original patient tumor cell population and architecture, histopathology, genomic and transcriptomic landscape, and heterogeneity. Furthermore, PDX response to anticancer drugs in mice has been closely correlated to the original patient's susceptibility to these treatments in the clinic. Several PDXs have been established and have undergone critical in-depth characterization at the cellular and molecular level across multiple PCa tumor subtypes representing both primary and metastatic patient tumors and their inherent levels of androgen responsiveness and/or treatment resistance, including androgen-sensitive, castration resistant, and neuroendocrine PCa. Multiple PDX networks and repositories have been generated for the collaborative and shared use of these vital translational cancer tools. Here we describe the creation of a PDX maintenance colony from an established well-characterized PDX, best practice for PDX maintenance in mice, and their subsequent application in preclinical drug testing. This chapter aims to serve as a go to resource for the preparation and adoption of PCa PDX models in the research laboratory and for their use as a valuable preclinical platform for translational research and therapeutic agent development.

Targeting the αVβ3/NgR2 pathway in neuroendocrine prostate cancer

Highly aggressive, metastatic, neuroendocrine prostate cancer, which typically develops from prostate cancer cells acquiring resistance to androgen deprivation therapy, is associated with limited treatment options and hence poor prognosis. We have previously demonstrated that the αVβ3 integrin is over-expressed in neuroendocrine prostate cancer. We now show that LM609, a monoclonal antibody that specifically targets the human αVβ3 integrin, hinders the growth of neuroendocrine prostate cancer patient-derived xenografts in vivo. Our group has recently identified a novel αVβ3 integrin binding partner, NgR2, responsible for regulating the expression of neuroendocrine markers and for inducing neuroendocrine differentiation in prostate cancer cells. Through in vitro functional assays, we here demonstrate that NgR2 is crucial in promoting cell adhesion to αVβ3 ligands. Moreover, we describe for the first time co-fractionation of αVβ3 integrin and NgR2 in small extracellular vesicles derived from metastatic prostate cancer patients’ plasma. These prostate cancer patient-derived small extracellular vesicles have a functional impact on human monocytes, increasing their adhesion to fibronectin. The monocytes incubated with small extracellular vesicles do not show an associated change in conventional polarization marker expression and appear to be in an early stage that may be defined as “adhesion competent”. Overall, these findings allow us to better understand integrin-directed signaling and cell-cell communication during cancer progression. Furthermore, our results pave the way for new diagnostic and therapeutic perspectives for patients affected by neuroendocrine prostate cancer.

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.

Integrin-targeting disulfide-crosslinked micellar docetaxel eradicates lung and prostate cancer patient-derived xenografts

Actively targeted nanomedicines though conceptually attractive for tumor therapy are extremely hard to realize due to problems of premature drug leakage, excessive liver accretion, inadequate tumor uptake, and/or retarded drug release inside tumor cells. Here, we systemically studied the influence of disulfide crosslinking on the in vitro and in vivo performance of integrin-targeting micellar docetaxel (t-MDTX). Of note, t-M5DTX with a high disulfide content was clearly advantageous in terms of stability, intracellular drug release, anti-tumor activity toward αVβ3-overexpressing A549 cells, blood circulation and therapeutic efficacy in orthotopic A549-luc lung tumor-bearing mice. t-MDTX induced extraordinary tumor targetability with tumor-to-normal tissue ratios of 1.7–8.3. Further studies indicated that t-M5DTX could effectively eradicate αVβ3-overexpressing lung and prostate cancer patient-derived xenografts (PDX), in which ca. 80% mice became tumor-free. This integrin-targeting disulfide-crosslinked micellar docetaxel emerges as a promising actively targeted nanoformulation for tumor therapy. Statement of significanceNanomedicines have a great potential in treating advanced tumor patients; however, their tumor-targeting ability and therapeutic efficacy remain unsatisfactory. In addition to PEGylation and ligand selection, particle size, stability and drug release behavior are also critical to their performance in vivo. In this paper, we find that small and cRGD-guided disulfide-crosslinked micellar docetaxel (t-MDTX) induces superior tumor uptake and retention but without increasing liver burden, leading to extraordinary selectivity and inhibition of αvβ3 overexpressing lung tumors. t-MDTX is further shown to effectively treat αvβ3-positive patient-derived tumor models, lending it a high potential for clinical translation.

Third generation quinoline-3-carboxamide transcriptional disrupter of HDAC4, HIF-1α, and MEF-2 signaling for metastatic castration-resistant prostate cancer.

The quinoline-3-carboxamide, Tasquinimod (TasQ), is orally active as a maintenance therapy with an on-target mechanism-of-action via allosteric binding to HDAC4. This prevents formation of the HDAC4/NCoR1/HDAC3 complex, disrupting HIF-1α transcriptional activation and repressing MEF-2 target genes needed for adaptive survival signaling in the compromised tumor micro environment. In phase 3 clinical testing against metastatic castration-resistant prostate cancer(mCRPC), TasQ (1 mg/day) increased time-to-progression, but not overall survival. TasQ analogs were chemically synthesized and tested for activity compared to the parental compound. These included HDAC4 enzymatic assays, qRT-PCR and western blot analyses of gene and protein expression following treatment, in vitro and in vivo efficacy against multiple prostate cancer models including PDXs, pharmacokinetic analyses,AHR binding and agonist assays, SPR analyses of binding to HDAC4 and NCoR1, RNAseq analysis of in vivo tumors, 3D endothelial sprouting assays, and a targeted kinase screen. Genetic knockout or knockdown controls were used when appropriate. Here, we document that, on this regimen (1 mg/day), TasQ blood levels are 10-fold lower than the optimal concentration (≥2 μM) needed for anticancer activity, suggesting higher daily doses are needed. Unfortunately, we also demonstrate that TasQ is an arylhydrocarbon receptor (AHR) agonist, which binds with an EC50 of 1 μM to produce unwanted off-target side effects. Therefore, we screened a library of TasQ analogsto maximize on-target versus off-target activity. Using this approach, we identified ESATA-20, which has ~10-fold lower AHR agonism and 5-fold greater potency against prostate cancer patient-derived xenografts. This increased therapeuticindex nominates ESATA-20 as a lead candidate forclinical development as an orally active third generation quinoline-3-carboxamide analog thatretains its on-target ability to disrupt HDAC4/HIF-1α/MEF-2-dependent adaptive survival signaling in the compromisedtumor microenvironment found in mCRPC.

RGD-directed 24nm micellar docetaxel enables elevated tumor-liver ratio, deep tumor penetration and potent suppression of solid tumors.

Nanomedicines while showing a great potential in improving the performance of chemotherapeutics like docetaxel (DTX) are distressed by a high liver deposition and poor tumor penetration, which might not only cause liver toxicity but also moderate therapeutic effect. Herein, we report that cRGD-directed 24nm disulfide-crosslinked micellar docetaxel (cRGD-MDTX) presents low liver accumulation, high tumor uptake, and deep tumor penetration, leading to the potent suppression of different solid tumors. cRGD-MDTX was optimized with a cRGD density of 4% and DTX loading of 10wt%. Interestingly, cRGD-MDTX enabled an extraordinary tumor-liver ratio of 2.8/1 with a DTX uptake of 8.3 %ID/g in αvβ3 over-expressing PC3 prostate tumor. The therapeutic studies demonstrated striking antitumor effects of cRGD-MDTX toward PC3 prostate tumor, prostate cancer patient-derived xenografts (PDX), orthotopic A549-Luc lung cancer and orthotopic SKOV3-Luc ovarian tumor models, in which tumor growth was effectually inhibited and 6-8 times better improvement of median survival time over free DTX was observed. This small disulfide-crosslinked micellar drug capable of relegating liver deposition opens a new avenue to nanomedicines for targeted therapy.

Influence of ADT on B7-H3 expression during CRPC progression from hormone-naïve prostate cancer.

Androgen deprivation therapy (ADT) is the standard care for advanced prostate cancer (PCa) patients. Unfortunately, although tumors respond well initially, they enter dormancy and eventually progress to fatal/incurable castration-resistant prostate cancer (CRPC). B7-H3 is a promising new target for PCa immunotherapy. CD276 (B7-H3) gene has a presumptive androgen receptor (AR) binding site, suggesting potential AR regulation. However, the relationship between B7-H3 and AR is controversial. Meanwhile, the expression pattern of B7-H3 following ADT and during CRPC progression is largely unknown, but critically important for identifying patients and determining the optimal timing of B7-H3 targeting immunotherapy. In this study, we performed a longitudinal study using our unique PCa patient-derived xenograft (PDX) models and assessed B7-H3 expression during post-ADT disease progression. We further validated our findings at the clinical level in PCa patient samples. We found that B7-H3 expression was negatively regulated by AR during the early phase of ADT treatment, but positively associated with PCa proliferation during the remainder of disease progression. Our findings suggest its use as a biomarker for diagnosis, prognosis, and ADT treatment response, and the potential of combining ADT and B7-H3 targeting immunotherapy for hormone-naïve PCa treatment to prevent fatal CRPC relapse.

Abstract A077: MicroRNA drivers of resistance to androgen deprivation therapy in prostate cancer

Abstract INTRODUCTION: Prostate cancer is the most prevalent malignancy affecting Western males. It is initially an androgen-dependent disease: androgens bind to the androgen receptor and drive expression of genes that promote proliferation and evasion of apoptosis. Despite reduced androgen dependence in advanced prostate cancer, androgen receptor signalling remains a key driver of growth. Androgen deprivation therapy (ADT) is therefore a first line treatment approach and works well initially, but resistance inevitably develops. Abiraterone and Enzalutamide are drugs widely used in ADT and are androgen synthesis and androgen receptor signalling inhibitors respectively. The shortage of other treatment options means acquired resistance to these drugs is a major clinical problem. MicroRNAs (miRs) are important mediators of post-transcriptional gene regulation and show altered expression in cancer. Several have been linked to the development of resistance to ADT. Manipulation of such miRs may be a pathway to breakthrough treatments for advanced prostate cancer. This study aimed to validate ADT resistance-implicated miRs and their clinically relevant targets. MATERIALS AND METHODS: Small RNA-sequencing of Abiraterone- and Enzalutamide-resistant C42 prostate cancer cells identified subsets of miRs dysregulated as compared to parental cells. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) was used to validate altered expression of candidate ADT resistance-implicated miRs 195-5p, 497-5p and 29a-5p in ADT-resistant and -responsive prostate cancer cell lines, patient-derived xenografts (PDXs) and primary prostate cancer explants. RESULTS AND DISCUSSION: This study suggests a possible role for miR-497-5p in the development of ADT resistance in prostate cancer. MiR-497-5p expression was increased in ADT-resistant versus ADT-responsive prostate cancer cells. Importantly, miR-497-5p expression was also increased in Enzalutamide-treated, castrated (ADT-mimicking) PDXs versus intact PDXs. MiR-195-5p was also elevated in ADT-resistant versus -responsive prostate cancer cells, while there was a drop in miR-29a-5p expression. Candidate clinically relevant targets of miR-497-5p in prostate cancer were identified by mining AGO-PAR-CLIP-seq data sets and may include AVL9 and FZD6. CONCLUSION: In summary, this study identified microRNAs that are implicated in prostate cancer resistance to androgen deprivation therapy and could represent novel therapeutic targets for advanced disease. Citation Format: Philippa C. Saunders, Claire Fletcher. MicroRNA drivers of resistance to androgen deprivation therapy in 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 A077.

Abstract IA003: Evolution of AR gene structure during prostate cancer progression

Abstract Castration-resistant prostate cancer (CRPC) emerges when endocrine therapies are no longer effective at suppressing activity of the androgen receptor (AR) transcription factor. Our laboratory has implemented DNA sequencing approaches to identify genomic mechanisms contributing to CRPC in prostate cancer cell lines, patient-derived xenografts (PDXs), and clinical specimens. These approaches have revealed AR as one of the most frequently-rearranged genes in CRPC. However the patterns of AR gene rearrangements observed between patients and even between anatomically distinct metastases within a single patient are remarkably complex and heterogeneous. To address this complexity, we analyzed a cohort of prostate cancer specimens using linked-read DNA-sequencing. This approach found that a subset of clinical CRPC metastases had accumulated complex AR rearrangements in conjunction with very high AR copy number, a phenomenon that also occurred in PDX models grown under selective pressure of castration. Using orthogonal approaches of optical genome mapping and fluorescence in situ hybridization, we found that AR residing on extrachromosomal circular DNA (eccDNA) was an underlying mechanism. This study identifies eccDNA as an engine for accumulation of AR gene copy number and structural complexity during CRPC progression. Citation Format: Scott M Dehm. Evolution of AR gene structure during prostate cancer progression [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 IA003.

Viability Analysis and High-Content Live-Cell Imaging for Drug Testing in Prostate Cancer Xenograft-Derived Organoids

Tumor organoids have been pushed forward as advanced model systems for in vitro oncology drug testing, with the eventual goal to direct personalized cancer treatments. However, drug testing efforts suffer from a large variation in experimental conditions for organoid culturing and organoid treatment. Moreover, most drug tests are restricted to whole-well viability as the sole read-out, thereby losing important information about key biological aspects that might be impacted due to the use of administered drugs. These bulk read-outs also discard potential inter-organoid heterogeneity in drug responses. To tackle these issues, we developed a systematic approach for processing organoids from prostate cancer (PCa) patient-derived xenografts (PDXs) for viability-based drug testing and identified essential conditions and quality checks for consistent results. In addition, we generated an imaging-based drug testing procedure using high-content fluorescence microscopy in living PCa organoids to detect various modalities of cell death. Individual organoids and cell nuclei in organoids were segmented and quantified using a dye combination of Hoechst 33342, propidium iodide and Caspase 3/7 Green, allowing the identification of cytostatic and cytotoxic treatment effects. Our procedures provide important insights into the mechanistic actions of tested drugs. Moreover, these methods can be adapted for tumor organoids originating from other cancer types to increase organoid-based drug test validity, and ultimately, accelerate clinical implementation.

Abstract LB298: Molecular phenotype classification of metastatic prostate cancer by cell-free DNA methylation analysis

Abstract Introduction: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease which can be classified into clinically relevant subtypes based on the expression of transcription factors (TF), such as the androgen receptor (AR) and neuroendocrine markers. Neuroendocrine prostate cancer (NEPC), characterized by gain of stem-like and neuroendocrine features and lack of AR expression is a clinically aggressive variant. Due to the absence of adequate biomarkers, NEPC is usually detected at a very advanced stage. There is mounting evidence that molecular subtype changes seen in NEPC are enforced by widespread epigenetic alterations, in particular DNA methylation changes. In this study, we aim to devise a novel DNA methylation-based assay for molecular subtyping and disease monitoring from cell-free DNA (cfDNA). Methods: We analyzed genome wide methylation patterns in 60 prostate cancer patient-derived xenograft (PDX) and 133 mCRPC tumors using array- and sequencing-based assays. We integrated DNA methylation with TF cistrome data to determine the landscape of methylation alterations at key lineage TF binding sites (TFBS). A linear regression model was trained on low-pass Enzymatic Methyl-Seq (EM-seq) cfDNA data derived from PDXs to identify molecular subtype specific DNA methylation changes at these TFBS. The model performance was optimized with in silico admixture experiments. This model was then used to discern tumor molecular phenotypes from cfDNA in three independent cohorts of mCRPC patients using low-pass whole genome bisulfite sequencing and EM-seq. Results: We observed a strong association between TFBS methylation and TF expression. For lineage specific TFs such as AR and ASCL1, we identified core sets of TFBSs whose differential methylation allowed for accurate assay-independent molecular subtype classification in tumor tissues. Applying an optimized quantitative model to mCRPC patients who underwent comprehensive tissue sampling by rapid autopsy we observed perfect subtype prediction from both tissue samples and cfDNA (AUC=1). A similar analytical performance was observed in additional clinical mCRPC cohorts with cfDNA. Conclusions: We show that methylation patterns at TFBSs can determine TF activity and can be used to classify molecular subtypes from both tumor tissue and cfDNA. For prostate cancer, we demonstrate that this approach can accurately detect NEPC by cost-effective low-pass EM-seq. More broadly, this study provides a novel analysis framework for robustly assessing molecular tumor phenotypes in cfDNA with applications in solid and liquid tumor diagnostics. Citation Format: Mohamed Adil, Brian Hanratty, Pallabi Mustafi, Ilsa Coleman, Radhika Patel, Anna-Lisa Doebley, Robert Patton, Eden Cruikshank, Patricia Galipeau, Ruth Dumpit, Martine Roudier, Jin-Yih Low, Navonil De Sarkar, Robert Montgomery, Eva Corey, Colm Morrissey, Peter Nelson, Gavin Ha, Michael Haffner. Molecular phenotype classification of metastatic prostate cancer by cell-free DNA methylation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB298.

Abstract 1789: Pre-clinical studies to advance anti-ROR1 CAR-T cell therapy for metastatic prostate cancer

Abstract Background: Alterations in WNT signaling are frequently associated with tumorigenesis and metastasis in many cancers including prostate cancer (PCa). The Wnt ligand, WNT5A, is required for normal prostate gland development and is increased in bone metastatic, castration resistant PCa (CRPC) patients. WNT5A signaling is mediated in part through ROR1, a non-canonical Wnt receptor and fetal oncoprotein for which the therapeutic inhibitory antibody, Zilovertamab, has been developed. Its safety has been clinically proven in trials for chronic lymphocytic leukemia (CLL) and metastatic breast cancer. We sought to investigate Zilovertamab-based anti-ROR1 therapies for metastatic prostate cancer.Our Hypothesis is that WNT5A may activate a stem-cell-like program via ROR1 which leads to therapy resistance in metastatic prostate cancer. The anti-ROR1 biologic, Zilovertamab, may inhibit this mechanism of resistance. The expression of ROR1 on CRPC and NEPC tumors and its lack of expression on normal adult tissues makes it a promising CART cell target. Methods: We used the patient-derived xenograft (PDX), PCSD13, small cell bone metastatic prostate cancer model and the neuroendocrine PCa cell lines, PC3 and DU145, to test the effect of Zilovertamab and Zilovertamab-CART cells in vitro using the real time cell viability, proliferation, and cell cycle tracking assays in an Incucyte S3. We used in vivo bioluminescence and tumor caliper measurements to monitor effects in vivo. Results: Studies using RNASeq, qRT-PCR, FACS and Westerns showed high expression of ROR1 in PC3, DU145, and in PCSD13. CRISPR-Cas9 Knock out of ROR1 in PC3 and DU145 cells showed increased inhibition of proliferation at lower docetaxel concentrations. Treatment of PCSD13 PDX in vivo with Zilovertamab increased docetaxel-mediated tumor growth inhibition. Mice bearing PC3 xenografts injected intravenously with Zilovertamab anti-ROR1 CAR-T cells showed durable, tumor ablation in 67% of mice compared to 22% of mice injected with activated T cells from the same donor and 0% of untreated mice. Survival of mice at Day 70 injected with CART was 78% compared to 0% of mice injected with control donor T cells and 0% of untreated mice. Zilovertamab antibody could inhibit Zilovertamab CART cell tumor cell killing. Conclusions: ROR1 was expressed at high levels on castration resistant small cell PCa and neuroendocrine PCa cell lines and PDX models. Zilovertamab synergized with docetaxel to inhibit tumor growth in patient derived xenograft in vivo and in organoid cultures. Zilovertamab-based CART cells durably eradicated ROR1+ prostate cancer xenograft tumors. These studies supported the recently launched Phase 1b clinical trial of Zilovertamab plus docetaxel in metastatic CRPC patients. Clinical development of GMP Zilovertamab CART cells for a clinical trial in CLL is in progress and may lead to rapid progression to a clinical trial for metastatic CRPC and NEPC. Citation Format: Christina A.M. Jamieson, Jamillah Murtadha, Christopher S. Oh, Michelle Muldong, Evodie Koutouan, JOngwook Kim, Niloofar Etemadfard, Hae Soo Choo, Navyaa Sinha, Sanghee Lee, Christina Wu, Gabriel Pineda, Kathleen Lennon, Karl Willert, Catriona H. Jamieson, Terry Gaasterland, Rana Mckay, Christopher J. Kane, Thomas J. Kipps, Anna A. Kulidjian, Nicholas A. Cacalano, Charles Prussak. Pre-clinical studies to advance anti-ROR1 CAR-T cell therapy for metastatic prostate cancer [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 1789.