Preclinical Models Of Prostate Cancer Research Articles

A Radiotracer for Molecular Imaging and Therapy of Gastrin-Releasing Peptide Receptor-Positive Prostate Cancer.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in many solid malignancies, particularly in prostate and breast cancers, among others. We synthesized ProBOMB2, a novel bombesin derivative radiolabeled with 68Ga and 177Lu, and evaluated its ability to target GRPR in a preclinical model of human prostate cancer. Methods: ProBOMB2 was synthesized in solid phase using fluorenylmethoxycarbonyl chemistry. The chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid was coupled to the N terminus and separated from the GRPR-targeting sequence by a cationic 4-amino-(1-carboxymethyl)-piperidine spacer. Binding affinity for both human and murine GRPR was determined using a cell-based competition assay, whereas a calcium efflux assay was used to measure the agonist and antagonist properties of the derivatives. ProBOMB2 was radiolabeled with 177Lu and 68Ga. SPECT and PET imaging and biodistribution studies were conducted using male immunocompromised mice bearing GRPR-positive PC-3 human prostate cancer xenografts. Results: Ga-ProBOMB2 and Lu-ProBOMB2 bound to PC-3 cells with an inhibition constant of 4.58 ± 0.67 and 7.29 ± 1.73 nM, respectively. 68Ga-ProBOMB2 and 177Lu-ProBOMB2 were radiolabeled with a radiochemical purity greater than 95%. Both radiotracers were excreted primarily via the renal pathway. PET images of PC-3 tumor xenografts were visualized with excellent contrast at 1 and 2 h after injection with 68Ga-ProBOMB2, and there was very low off-target organ accumulation. 177Lu-ProBOMB2 enabled clear visualization of PC-3 tumor xenografts by SPECT imaging at 1, 4, and 24 h after injection 177Lu-ProBOMB2 displayed higher tumor uptake than 68Ga-ProBOMB2 at 1 h after injection. 177Lu-ProBOMB2 tumor uptake at 1, 4, and 24 h after injection was 14.9 ± 3.1, 4.8 ± 2.1, and 1.7 ± 0.3 percentage injected dose per gram of tissue, respectively. Conclusion: 68Ga-ProBOMB2 and 177Lu-ProBOMB2 are promising radiotracers with limited pancreas uptake, good tumor uptake, and favorable pharmacokinetics for imaging and therapy of GRPR-expressing tumors.

Abstract 1781: Correlates of androgen deprivation and gut microbiome in mouse Pten-deficient prostate cancer

Abstract Androgen receptor (AR) signaling plays a key role in the progression of prostate cancer and androgen deprivation therapy (ADT) is one the standard of care for men with advanced prostate cancer. However, AR also exerts effects on androgen responsive tissues and accumulating data indicates that AR is implicated in influencing gut flora. Commensal bacteria are essential for maintaining a healthy immune system which plays a major role in cancer surveillance. Moreover, cancer is known to promote commensal dysbiosis which can further impair immune function. Here, we investigate associations between commensal fecal bacteria and androgen deprivation in an immunocompetent transgenic mouse model of Pten-deficient prostate cancer. 16S rRNA amplicon sequencing was used to comprehensive profile and compare the fecal microbiomes of bearing conditional Pten-knockout mice with those of mice 5 weeks after surgical castration. Tumor burden in surgically castrated mice was significantly lower compared to that of intact mice, 57.45%, P<0.001. With regards to fecal microbial composition, ADT via surgical castration enhanced alpha diversity (Shannon index, P=0.006). Moreover, microbial communities differed between castrated and intact mice (PERMANOVA, P=0.003, NMDS stress=0.042). Notably, an individual mouse with a strong response to ADT showed marked differences in fecal microbe composition compared to other mice in the cohort. Taxa associated with ADT included Dorea, Oscillospora, Sutturella, Dehalobacterium, Akkermansia muciniphila, and Flexispira while Prevoltella, Allobacalum, Ruminococcus flavefaciens, Candidatus Arthromitus and Bacteroides. Dehalobacterium, Coprococcus and Ruminococcus gnavus were more abundant in fecal samples from castrated, low tumor burden mouse. Overall, Bacteriodes and Parabacteriodes were positively correlated to tumor burden. Taxon enrichment analysis (TSEA) revealed functional enrichments of lipid translocation, carbohydrate transport, amino acid transport and proteoglycans in cancer in castrated mice, whereas, enrichment of low-density lipoprotein particle binding, extracellular matrix assembly, inflammatory response to antigenic stimulus and ADP metabolic process. Functional profiles inferred from metagenomic 16S rRNA data revealed enrichment of glyoxylate and dicarboxylate metabolism, carbon metabolism and geraniol metabolism in castrated mice. In intact mice, lipopolysaccharide biosynthesis, streptomycin biosynthesis and amino sugar and nucleotide sugar metabolism were enriched. Notably, lower tumor burden was associated with porphyrin and chlorophyll metabolism, propanoate metabolism, and valine, leucine and isoleucine degradation. This study models ADT in a preclinical model of prostate cancer and provides a broad characterization of the gut microbiome and its association to androgen deprivation. Citation Format: Marco A. De Velasco, Kazuko Sakai, Yurie Kura, Eri Banno, Naomi Ando, Noriko Sako, Nobutaka Shimizu, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Correlates of androgen deprivation and gut microbiome in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1781.

Abstract 2929: Comparative analysis of circulating human tumor DNA and bioluminescent imaging in monitoring tumor burden and therapeutic response of ionizing radiation in intratibial human prostate tumor xenografts

Abstract Purpose/Objective: Pre-clinical models of prostate cancer (PCa) metastases provide critical insight into tumor biology and therapeutic response. Current models require sophisticated reporter transgene models and/or expensive tools to noninvasively monitor tumors in metastatic sites, such as bone. Recent developments in the measurement of circulating tumor DNA (ctDNA) provide an alternative method to noninvasively assess tumor burden at a low cost. Here we present a comparative analysis of human PCa xenograft tumor growth and therapeutic response by simultaneous measurement of ctDNA and bioluminescence. Materials and methods: Male athymic nude mice were used for intra-tibial (n=10) modeling. LNCaP-CMV-Luc cells were implanted in the tibial medullary canal. Tumor burden was evaluated at pre and post-IR (focal exposure 6 Gy on day 28 post-implantation) time-points by in vivo bioluminescent imaging and quantitative real-time polymerase chain reaction (qPCR) for human LINE1/Alu DNA. qPCR was performed on plasma-derived DNA consecutively on the same day as bioluminescence. Results: Human genomic DNA quantification was sensitive to 0.1 pg and linear by hAlu (R2=0.98, for 0.1pg-150ng) and LINE DNA (R2=0.99, for 0.1pg-150ng) qPCR. hAlu DNA qPCR produces a much lower Ct value when compared to LINE1; possibly due to its higher copies in the human genome (Ct value 28 versus 22/0.1 pg). Following tumor inoculation, ctDNA level and tumor bioluminescence were strongly and significantly correlated over time (LINE1, r=0.65 and Alu, r=0.59, p<0.002). Following radiation, we observed an immediate (day 32) but transient decrease in the levels of ctDNA and bioluminescence, followed by a gradual increase due to tumor relapse. The correlation between ctDNA level and bioluminescence was not maintained, but responses followed a similar trend. Conclusions: In the present study, we report that the volume of human PCa xenografts can be accurately measured in the intra-tibial compartments of athymic nude mice by qPCR of circulating human Alu and LINE1 elements. Plasma-derived ctDNA levels highly correlated with tumor bioluminescence over time. Tumor directed radiation therapy exhibited slightly differential responses in ctDNA level and tumor bioluminescence, but overall both measurements followed a similar trend. Citation Format: Alok Mishra, Kenji Zennami, Esteban Velarde, Jonathan B. Coulter, Srinivasan Yegnasubramanian, Shawn E. Lupold, Theodore L. DeWeese. Comparative analysis of circulating human tumor DNA and bioluminescent imaging in monitoring tumor burden and therapeutic response of ionizing radiation in intratibial human prostate tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2929.

Abstract 1420: GR antagonist ORIC-101 overcomes GR-mediated resistance to the combination of AR and AKT inhibition in preclinical prostate cancer cell lines

Abstract Androgen deprivation therapy (ADT) with or without antiandrogens such as enzalutamide is the mainstay of treatment in advanced prostate cancer (PC). However, new therapeutic strategies are needed for patients who relapse on antiandrogen therapy. The glucocorticoid receptor (GR) has been implicated as a potential antiandrogen bypass mechanism. We previously showed that ORIC-101, a potent and selective, orally bioavailable small molecule GR antagonist, reverses GR-mediated resistance to enzalutamide in preclinical PC models. In the clinic, we are evaluating ORIC-101 in combination with enzalutamide in patients with metastatic prostate cancer (NCT04033328). Activation of the PI3K/AKT pathway through PTEN loss is a poor prognostic factor and another potential resistance mechanism to anti-hormone directed therapies. Reciprocal crosstalk between androgen signaling and PI3K/AKT pathway activation forms the basis for the clinical development of AKT inhibitors (AKTi) in combination with ADT or antiandrogens (NCT03072238, NCT04087174). In this study, we tested preclinically whether activated GR confers resistance to the combination of AKT inhibitors with enzalutamide and whether co-treatment with ORIC-101 reverses GR-mediated resistance to an enzalutamide/AKTi doublet. We first evaluated the effects of enzalutamide plus AKTi treatment on GR levels, GR target genes and activity in three prostate cancer cell lines: PTEN-null line LNCaP and PTEN-wildtype lines VCaP and CWR22PC. We tested three AKT inhibitors: the ATP-competitive inhibitors ipatasertib (GDC-0068) and capivasertib (AZD5363), in addition to the allosteric inhibitor MK2206, in CSS media and 10% FBS, with or without supplementation of the synthetic glucocorticoid, dexamethasone. An increase in the protein and mRNA levels of GR and GR target genes was observed after 7 days of enzalutamide treatment. In the AKTi plus enzalutamide setting, AKT inhibition did not block the enzalutamide-mediated GR upregulation in either PTEN-null or PTEN-wildtype cells. We next evaluated whether increased GR activity mediates resistance to the enzalutamide/AKTi doublet with ipatasertib or capivasertib, and whether this resistance can be reversed with ORIC-101. GR activation with dexamethasone promoted tumor cell growth and androgen-regulated gene expression in the presence of enzalutamide and ipatasertib or capivasertib, using a 21-day proliferation assay in CWR22PC cells. ORIC-101 was able to reverse these dexamethasone-induced effects, confirmed by reduced secreted PSA levels and cell number measured at end of study. These findings indicate that GR upregulation and activation, an established resistance mechanism for antiandrogens, may drive resistance when combined with an AKT inhibitor, and the GR antagonist ORIC-101 is able to overcome this resistance and restore antitumor activity. Citation Format: Shravani Barkund, Haiying Zhou, Lori S. Friedman, Anneleen Daemen. GR antagonist ORIC-101 overcomes GR-mediated resistance to the combination of AR and AKT inhibition in preclinical prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1420.

Abstract 42: Treatment-induced embryonic diapause-like adaptation through suppression of Myc activity as mediator of drug persistence in cancer

Abstract Chemo-persistent residual tumors are a major barrier for curative cancer therapy and provide a reservoir of cancer cells for eventual relapse. This clinically critical tumor cell population is poorly understood and lacks faithful in vitro models. We compared the transcriptional profiles of paired samples from patient with solid tumors or hematological neoplasias at the stage of post-treatment residual tumors or minimal residual disease vs. at their respective baselines. For a large proportion of patients, post-treatment tumor cells had a molecular signature that resembled that of embryonic diapause, a dormant stage of suspended development in early mammalian embryos triggered by stress and induced by suppression of Myc activity and overall biosynthesis. This Myc-inactivated molecular program was acquired during treatment and not pre-existing at baseline. Remarkably, baseline tumors with multiple MYC copy numbers maintained their MYC amplification in the post-treatment persistent tumor cells but suppressed Myc activity. We simulated this cancer cell state using preclinical models of breast and prostate cancer and multiple myeloma, including patient-derived cancer organoids and PDX models. Parallel approaches of genome sequencing and DNA barcode-mediated clonal tracking in vitro and in vivo independently indicated that the residual cancer cell subpopulations that persisted after treatment with common chemotherapeutic classes were not driven by rare pre-existing clones or de novo genetic events. Transcriptional analysis of the treatment-persistent cancer cells in 3D organoid cultures and in xenograft/PDX models indicated a diapause-like transcriptional adaptation with inactivated Myc, similar to that in patients. We functionally examined the role of Myc suppression in in vitro models of breast cancer and multiple myeloma using genetic (CRISPR/Cas9 editing and interference) and pharmacological (inhibition of Myc transcriptional co-activator Brd4) approaches. Suppression of Myc in cancer cells induced an embryonic diapause-like molecular profile and attenuated the acute chemotherapeutic cytotoxicity. Myc-inactivated cancer cells had reduced apoptotic priming and maintained low redox stress even in the presence of cytotoxic agents, which may contribute to cell survival during treatment. High-throughput screening of chemo-persistent cells revealed that inhibitors of CDK9 could revert the biosynthetic pause, reactivate Myc, attenuate the diapause-like state, and re-sensitize the residual cancer cells to chemotherapy. Overall, our study shows that cancer cells can co-opt the stress survival mechanism of embryonic diapause by dynamically suppressing Myc to enter transient drug-refractory dormancy. Citation Format: Eugen Dhimolea, Ricardo De Matos Simoes, Dhvanir Kansara, Caroline Vilas, Aziz Al'Khafaji, Juliette Bouyssou, Aedin Culhane, Constantine S. Mitsiades. Treatment-induced embryonic diapause-like adaptation through suppression of Myc activity as mediator of drug persistence in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 42.

Abstract 1568: A2aR inhibition enhances the antitumor activity of CTLA4 blockade in mouse Pten-deficient prostate cancer

Abstract Extracellular adenosine impairs immune function in tumors and limits the efficacy of anti-CTAL4 immune checkpoint blockade. We previously showed that adenosine 2A receptor (A2aR) blockade with AZD4635 moderately inhibited tumor growth, in part by improving antigen presentation and cytotoxic T cell (CTL) activity in mouse Pten-deficient prostate cancer. Here, we continue to investigate the antitumor activity of AZD4635 and its effects when combined with CTLA4 blockade (aCTLA4) in preclinical models of Pten-deficient prostate cancer. Prostate tumors from Pten conditional knockout (KO) mice treated with aCTLA4 for five days had increased expression levels of adenosinergic genes. Furthermore, gene expression, flow cytometric and IHC analyses showed increased T regulatory cell differentiation and accumulation that was offset with the addition of AZD4635. Moreover, expression levels of genes associated with Th 1 cell differentiation and T cell-mediated cytotoxicity were increased in AZD4635/aCTLA4 treated tumors. Adding AZD4635 to a four-week regimen of aCTLA4 therapy improved the antitumor response by two-fold in an early-stage intervention model of Pten-deficient prostate cancer. Androgen deprivation (AD) has the potential to promote T cell infiltration in prostate tumors and using the AZD4635/aCTLA4 treatment combination as neoadjuvant therapy to AD via surgical castration led to a greater antitumor response. The antitumor activity of this treatment combination was further examined and confirmed in a subcutaneous syngeneic genome-derived allograft model using tumor-bearing conditional Pten-deficient knockout mice grafted with Pten-deficient CRPC tumor fragments. In a Pten/Trp53 conditional double knockout (DKO) mouse model of advanced prostate cancer, the AZD4635/aCTLA4 treatment combination did not improve overall survival rates. However, when AZD4635/aCTLA4 was used as neoadjuvant therapy to AR inhibition with the anti-androgen apalutamide, it improved median survival time from 21 days in monotherapy treated mice to 34 days in AZD4635/aCTLA4 treated mice. These results provide preclinical evidence to support the rational combination of A2AR blockade with AZD4635 and aCTLA4 immune checkpoint inhibition for PTEN-deficient prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Alwin Schuller, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. A2aR inhibition enhances the antitumor activity of CTLA4 blockade in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1568.

Abstract 1438: Acute immune responses to apalutamide in mouse Pten-deficient prostate cancer

Abstract The androgen receptor (AR) is a major therapeutic target for human prostate cancer and therapies targeting the AR pathway also impact local and systemic immunity. However, the influences over host immune function vary depending on the type of AR treatment. Androgen deprivation (AD) can improve immune cell infiltration by enhancing T cell trafficking, but it also attracts immunosuppressive myeloid cells and regulatory T cells. We previously showed that chronic dosing of apalutamide, an oral nonsteroidal AR antagonist, was effective in suppressing tumor growth in a preclinical mouse models of Pten-deficient prostate cancer and also changed the composition of the tumor immune microenvironment improving cytotoxic T cell activity. Moreover, we also showed that using AD as neoadjuvant therapy improved the antitumor activity and immune responses of combined JAK/PD-L1 blockade, whereas administered as concurrent or adjuvant therapy was ineffective. Our aim in this study is to determine the potential of using apalutamide in as combination therapy with immunotherapy and immune modulating agents. For this we have characterized the acute immune modulatory activity of apalutamide on mouse Pten-deficient and compared its activity against that of AD. Conditional prostate specific Pten/Trp53 double knockout mice were orchidectomized or treated with apalutamide (30 mg/kg/D) for five days. Tumor samples and lymphoid organs were collected and processed for immune profiling using a qRT-PCR focused panel, flow cytometry and/or immunohistochemistry. Prostates from orchidectomized mice tended to be smaller however there was no decrease in size for apalutamide treated mice. Notably, thymuses were significantly enlarged in both orchidectomized and apalutamide treated mice compared to control. Genes related to antigen presenting/dendritic cell (APC/DC) and activated DC were greater in apalutamide treated mice compared to vehicle treated or surgically castrated mice. This finding was consistent with an increased abundance of MHC-IIhi/CD80+ migratory DCs (CD11c+/XCR1+) in tumors of apalutamide treated mice. Tumor associated macrophage infiltration was also greater in apalutamide treated mice. Granulocyte infiltration was significantly greater after both castration and apalutamide therapy however the proportion of mature granulocytes was higher in castrated mice. T cell infiltration was comparable between orchidectomized and apalutamide treated mice, and regulatory T cell activity was increased in both groups compared to controls, but to a higher degree in orchidectomized mice. In summary, this provides critical data that will be essential when developing rational combinations with additional immunotherapies and immunomodulatory agents. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Kazuko Sakai, Nobutaka Shimizu, Eri Banno, Masahiro Nozawa, Kazuhiro Fujita, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Acute immune responses to apalutamide in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1438.

Abstract 1235: Selective androgen receptor degraders for the treatment of androgen receptor-positive, triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) is an aggressive breast cancer with shorter overall survival compared to other breast cancer types. One of the six molecularly-classified TNBC subtypes is the luminal androgen receptor subtype (LAR), which overexpresses androgen receptor (AR) and is dependent on AR for its growth. About 10-20% of TNBCs belong to the LAR subtype. Competitive AR antagonists, enzalutamide and bicalutamide, were effective in preclinical models of LAR TNBC and in clinical trials. This led us to hypothesize that potent selective AR degraders (SARDs), due to their ability to inhibit and degrade the AR, could provide a novel therapeutic approach for the treatment of LAR subtype of TNBC. Description: Western blots, cell line proliferation assays, and gene expression analyses were performed to evaluate novel small molecule SARDs. LAR TNBC cell lines and patient-derived xenografts (PDX) were utilized for in vivo evaluation of the SARDs. Once tumors grew to ~100-300 mm3, mice were randomized and treated orally for four weeks with vehicle, SARD UT-34, SARD UT-105, enzalutamide, or bicalutamide. Tumor volumes were measured twice weekly and tumors were collected at sacrifice for further analyses. Summary: SARDs bind to the N-terminus of the AR and have been characterized in preclinical advanced prostate cancer models. In this study, the SARDs were evaluated in preclinical models of LAR TNBC. Western blot for AR in LAR MDA-MB-453 cells demonstrated degradation of the AR protein by SARDs at low micromolar concentrations. Gene expression studies showed a complete inhibition of androgen-induced AR target gene transcription by the SARDs. Androgen-induced proliferation of MDA-MB-453 cells was inhibited by SARDs. MDA-MB-453 cells implanted subcutaneously in NOD SCID Gamma female mice grew robustly to 100-300 mm3 in 15-20 days. Treatment of tumor-bearing animals with the SARDs completely inhibited or regressed the tumors. Conclusion: These results support the findings that AR is the driver of MDA-MB-453 cell and tumor growth. SARDs with their unique mechanism of action may provide a new therapeutic option to women affected by the LAR subtype of TNBC. Disclosure: This work was partially supported by Oncternal Therapeutics and by an NCI supplement award to R01 (CA229164S1 to author RN). The SARD program has been licensed to Oncternal Therapeutics, Inc. by the University of Tennessee Research Foundation. Author RN is a consultant to Oncternal Therapeutics. Citation Format: Sarah Asemota, Kirsten Young Young, Suriyan Ponnusamy, Thirumagal Thiyagarajan, Dong-Jin Hwang, Yali He, James B. Breitmeyer, Gunnar F. Kaufmann, Duane D. Miller, Ramesh Narayanan. Selective androgen receptor degraders for the treatment of androgen receptor-positive, triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1235.

High-Throughput Imaging Assay for Drug Screening of 3D Prostate Cancer Organoids.

New treatments are required for advanced prostate cancer; however, there are fewer preclinical models of prostate cancer than other common tumor types to test candidate therapeutics. One opportunity to increase the scope of preclinical studies is to grow tissue from patient-derived xenografts (PDXs) as organoid cultures. Here we report a scalable pipeline for automated seeding, treatment and an analysis of the drug responses of prostate cancer organoids. We established organoid cultures from 5 PDXs with diverse phenotypes of prostate cancer, including castrate-sensitive and castrate-resistant disease, as well as adenocarcinoma and neuroendocrine pathology. We robotically embedded organoids in Matrigel in 384-well plates and monitored growth via brightfield microscopy before treatment with poly ADP-ribose polymerase inhibitors or a compound library. Independent readouts including metabolic activity and live-cell imaging–based features provided robust measures of organoid growth and complementary ways of assessing drug efficacy. Single organoid analyses enabled in-depth assessment of morphological differences between patients and within organoid populations and revealed that larger organoids had more striking changes in morphology and composition after drug treatment. By increasing the scale and scope of organoid experiments, this automated assay complements other patient-derived models and will expedite preclinical testing of new treatments for prostate cancer.

P0423 - Patient-derived preclinical models of castration resistant prostate cancer: Xenografts and PDX-derived organoids to recapitulate patients’ disease profiles

P0423 - Patient-derived preclinical models of castration resistant prostate cancer: Xenografts and PDX-derived organoids to recapitulate patients’ disease profiles

The Courage study: A first-in-human phase 1 study of the CBP/p300 inhibitor FT-7051 in men with metastatic castration-resistant prostate cancer.

TPS5085 Background: Prostate cancer is the second leading cause of cancer-related death among men in the U.S., largely due to metastatic disease that progresses despite hormonal therapy (tx). The role for androgen receptor (AR) signaling in prostate cancer and hormone tx resistance is well-established. CBP/p300 are essential co-activators of AR-mediated transcription. FT-7051 is an oral, potent, and selective inhibitor of CBP/p300 with activity in preclinical models of prostate cancer including models resistant to currently used AR inhibitors like enzalutamide. The Courage Study (NCT04575766) is a first-in-human, multicenter, phase 1, open-label study examining the safety, pharmacokinetics (PK), preliminary anti-tumor activity, and pharmacodynamics (PD) of FT-7051 for the treatment of men with metastatic castration-resistant prostate cancer (mCRPC) who have progressed despite prior tx and have been treated with at least one approved androgen receptor pathway inhibitor. The study will enroll up to 45 men with mCRPC at ̃8-15 US sites. Methods: The study employs a Bayesian optimal interval (BOIN) design with an accelerated titration. Patients (pts) will initially be enrolled in a dose level cohort size of 1 until a Grade 2 or higher toxicity occurs that is considered related to FT-7051 or the highest dose level is reached. Upon completion of the accelerated titration phase, subsequent cohorts will enroll 3-5 pts. Treatment: FT-7051 capsules will be administered on a 28 d cycle (21 d on / 7 d off) with Dose Levels -1 to 7 assigned per protocol using the BOIN design. Key inclusion criteria: Diagnosis of mCRPC with either adenocarcinoma or mixed histology AND rising PSA; previously failed at least one approved androgen receptor pathway inhibitor; ≥ 18 yrs of age; prior taxane chemotherapy permitted. Key exclusion criteria: Previous solid organ transplant, prior anticancer tx including prior tx with small molecules within 4 wks of first dose of study treatment, prior radiation tx within 4 wks prior to initiation of study treatment, prior androgen antagonist tx within 2 wks, prior radium-223 tx within 6 wks. Endpoints: Primary endpoints are to define the recommended phase 2 monotherapy dose of FT-7051 through assessments of DLTs, SAEs, clinically relevant AEs, and clinically relevant safety laboratory values. Key secondary endpoints include: PSA at 12 wks, time to PSA progression, time to radiographic progression, overall response rate, and plasma PK parameters. PD assessments of CBP/p300 inhibition in surrogate tissue, biomarker assessments in CTCs (AR, AR-v7), and peripheral blood are included. Duration: Pts will remain on study treatment until they are deemed to be no longer clinically benefiting (NLCB) by the treating Investigator or until unacceptable toxicity. Pt may be followed for survival for up to 24 months from last dose of study treatment. The first pt was dosed January 2021. Clinical trial information: NCT04575766.

Effect of Moderate and High Intensity Exercise on Hypoxia and Radiosensitivity in Tumor‐Bearing Rats

Background Prostate tumors typically have hypoxic regions that are resistant to traditional radiation therapy. Prior evidence from our laboratory has demonstrated improved perfusion and reduced hypoxia of prostate tumors with moderate intensity exercise training; such changes have potential to enhance radiation therapy. However, there is a dearth of evidence for the magnitude of alterations of intra-tumor hypoxia markers with exercise, as well as the ideal intensity of exercise for combating hypoxia in solid tumors. Using a pre-clinical orthotopic model of prostate cancer, we tested the hypothesis that exercise training at both moderate and high intensities would result in a decrease in hypoxia assessed via immunohistochemical analysis of hypoxic markers. Further, that enhanced radiotherapy responses would occur in exercise trained tumor bearing animals compared to sedentary counterparts. Methods Immunocompetent male Copenhagen rats aged 6 months (n=48) were injected orthotopically (ventral lobe of prostate) with 1x105 Dunning R-3327 AT-1 prostate adenocarcinoma cells. All animals were first acclimated to treadmill exercise and randomized into three groups: tumor bearing sedentary (TBS, n=20), moderate intensity training, (TBMIT, n=12), or high intensity training (TBHIT, n=16). After 7 days of recovery from surgery, both exercise groups began progressive exercise training on a motorized treadmill at either 20/min with a 5% incline, or 30m/min 15% incline for 10 minutes a day progressing to 60 min/day for TBMIT and TBHIT, respectively, for approximately 5 weeks. Immunohistochemical analysis and whole-body ionizing radiation (2 Gy) were performed at the end of the 5-week period of training followed by clonogenic cell survival assay to assess survival fraction. Results There were no significant differences (p>0.05) in tumor mass between groups (TBS 7.8±1.1; TBMIT 7.2±1.3; TBHIT 6.4±0.9 g). Overall expression levels of hypoxia inducible factor 1-alpha (HIF-1a, n=40) were significantly greater in the TBS group vs both the TBMIT and TBHIT (TBS 37.9±7.4; TBMIT 25.5±6.1; TBHIT 14.4±5.7%, p<0.05), with a trend for increased expression in TBMIT vs TBHIT (p=0.059). The survival fraction (n=8) was also significantly higher in TBS when compared to both TBMIT and TBHIT (TBS 39.9±5.3; TBMIT 23.2±3.2; TBHIT 20.7±4.9%, p<0.05). Conclusion This study suggests that compared to sedentary counterparts, both chronic moderate and high intensity exercise training modify the tumor microenvironment (decreased levels of HIF-1a) favorably. Given exercise therapy is becoming increasingly recommended to mitigate fatigue and increase quality of life in patients, a potential therapeutic component could add to the efficacy of exercise prescription in a clinical setting.

Development of a ghrelin receptor inverse agonist for positron emission tomography.

Imaging of Ghrelin receptors in vivo provides unique potential to gain deeper understanding on Ghrelin and its receptors in health and disease, in particular, in cancer. Ghrelin, an octanoylated 28-mer peptide hormone activates the constitutively active growth hormone secretagogue receptor type 1a (GHS-R1a) with nanomolar activity. We developed novel compounds, derived from the potent inverse agonist K-(D-1-Nal)-FwLL-NH2 but structurally varied by lysine conjugation with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), palmitic acid and/or diethylene glycol (PEG2) to allow radiolabeling and improve pharmacokinetics, respectively. All compounds were tested for receptor binding, potency and efficacy in vitro, for biodistribution and -kinetics in rats and in preclinical prostate cancer models on mice. Radiolabeling with Cu-64 and Ga-68 was successfully achieved. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiotracer were specifically accumulated by the GHS-R1a in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET. The radiotracer uptake was also partially blocked by K-(D-1-Nal)-FwLL-NH2 in stomach and thyroid. The presence of the GHS-R1a was also confirmed by immunohistology. In the arterial rat blood plasma, only the original compounds were found. The Cu-64 or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiolabeled inverse agonists turned out to be potent and safe. Due to their easy synthesis, high affinity, medium potency, metabolic stability, and the suitable pharmacokinetic profiles, they are excellent tools for imaging and quantitation of GHS-R1a expression in normal and cancer tissues by PET. These compounds can be used as novel biomarkers of the Ghrelin system in precision medicine.

Antitumor Activity of NLG207 (Formerly CRLX101) in Combination with Enzalutamide in Preclinical Prostate Cancer Models.

Effective treatments for patients with metastatic castration-resistant prostate cancer following disease progression on enzalutamide are currently an unmet clinical need. Simultaneous inhibition of the hypoxia-inducible factor (HIF)-1α and androgen receptor (AR) pathways has been previously shown to overcome enzalutamide resistance in vitro Combination treatment with NLG207, a nanoparticle-drug conjugate of camptothecin and inhibitor of HIF-1α, and enzalutamide was evaluated in preclinical prostate cancer models of enzalutamide resistance. The effect of NLG207 and enzalutamide on average tumor volume and tumor re-growth after 3 weeks of treatment was evaluated in vivo using the subcutaneous 22Rv1 xenograft and castrated subcutaneous VCaP xenograft models. Correlative assessments of antitumor activity were evaluated in vitro using cell proliferation and qPCR assays. NLG207 8 mg/kg alone and in combination with enzalutamide reduced average tumor volume by 93% after 3 weeks of treatment (P < 0.05) in comparison with vehicle control in the subcutaneous 22Rv1 xenograft model. Notably, the addition of NLG207 also enhanced the efficacy of enzalutamide alone in the castrated subcutaneous VCaP xenograft model, decreasing the median rate of tumor growth by 51% (P = 0.0001) in comparison with enzalutamide alone. In vitro assessments of cell proliferation and gene expression further demonstrated antitumor activity via AR-HIF-1α crosstalk inhibition. Combination treatment with NLG207 and enzalutamide was shown to be effective in preclinical prostate cancer models of enzalutamide resistance. Clinical investigation of this treatment combination is ongoing (NCT03531827).

The PSMA-targeting Half-life Extended BiTE Therapy AMG 160 has Potent Antitumor Activity in Preclinical Models of Metastatic Castration-resistant Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) remains a disease with high unmet medical need, as most patients do not achieve durable response with available treatments. Prostate-specific membrane antigen (PSMA) is a compelling target for mCRPC. It is highly expressed by primary and metastatic prostate cancer cells, with increased expression after progression on androgen deprivation therapy. We developed AMG 160, a half-life extended, bispecific T-cell engager immuno-oncology therapy that binds PSMA on prostate cancer cells and cluster of differentiation 3 on T cells for treatment of mCRPC. AMG 160 was evaluated in vitro and in mCRPC xenograft models. AMG 160 tolerability was assessed in nonhuman primates (NHP). AMG 160 activity as monotherapy and in combination with a PSMA-imaging agent, novel hormonal therapy, and immune checkpoint blockade was evaluated. AMG 160 induces potent, specific killing of PSMA-expressing prostate cancer cell lines in vitro, with half-maximal lysis of 6-42 pmol/L. In vivo, AMG 160 administered weekly at 0.2 mg/kg engages T cells administered systemically and promotes regression of established 22Rv-1 mCRPC xenograft tumors. AMG 160 is compatible with the imaging agent gallium 68-labeled PSMA-11, and shows enhanced cytotoxic activity when combined with enzalutamide or an anti-programmed death-1 antibody. AMG 160 exhibits an extended half-life and has an acceptable safety profile in NHPs. The preclinical characterization of AMG 160 highlights its potent antitumor activity in vitro and in vivo, and its potential for use with known diagnostic or therapeutic agents in mCRPC. These data support the ongoing clinical evaluation of AMG 160 in patients with mCRPC.See related commentary by Kamat et al., p. 2675.

Iron Induces Cell Death and Strengthens the Efficacy of Antiandrogen Therapy in Prostate Cancer Models.

In search of novel strategies to improve the outcome of advanced prostate cancer, we considered that prostate cancer cells rearrange iron homeostasis, favoring iron uptake and proliferation. We exploited this adaptation by exposing prostate cancer preclinical models to high-dose iron to induce toxicity and disrupt adaptation to androgen starvation. We analyzed markers of cell viability and mechanisms underlying iron toxicity in androgen receptor-positive VCaP and LNCaP, castration-resistant DU-145 and PC-3, and murine TRAMP-C2 cells treated with iron and/or the antiandrogen bicalutamide. We validated the results in vivo in VCaP and PC-3 xenografts and in TRAMP-C2 injected mice treated with iron and/or bicalutamide. Iron was toxic for all prostate cancer cells. In particular, VCaP, LNCaP, and TRAMP-C2 were highly iron sensitive. Toxicity was mediated by oxidative stress, which primarily affected lipids, promoting ferroptosis. In highly sensitive cells, iron additionally caused protein damage. High-basal iron content and oxidative status defined high iron sensitivity. Bicalutamide-iron combination exacerbated oxidative damage and cell death, triggering protein oxidation also in poorly iron-sensitive DU-145 and PC-3 cells.In vivo, iron reduced tumor growth in TRAMP-C2 and VCaP mice. In PC-3 xenografts, bicalutamide-iron combination caused protein oxidation and successfully impaired tumor expansion while single compounds were ineffective. Macrophages influenced body iron distribution but did not limit the iron effect on tumor expansion. Our models allow us to dissect the direct iron effect on cancer cells. We demonstrate the proof of principle that iron toxicity inhibits prostate cancer cell proliferation, proposing a novel tool to strengthen antiandrogen treatment efficacy.

Microparticle Encapsulation of a Prostate-targeted Biologic for the Treatment of Liver Metastases in a Preclinical Model of Castration-resistant Prostate Cancer

PRX302 is a highly potent, mutant bacterial pore-forming biologic protoxin engineered for selective activation by PSA, a serine protease expressed by benign and malignant prostate epithelial cells. Although being developed as a local therapy for benign prostatic hyperplasia and localized prostate cancer, PRX302 cannot be administered systemically as a treatment for metastatic disease due to binding to ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored proteins, which leads to poor accumulation within the tumor microenvironment. To overcome this limitation, poly-lactic-co-glycolic acid (PLGA) microparticles encapsulating the protoxin were developed, which are known to accumulate in the liver, a major site of metastasis for prostate cancer and other solid tumors. A highly sensitive and reproducible sandwich ELISA to quantify PRX302 released from microparticles was developed. Utilizing this assay, PRX302 release from different microparticle formulations was assessed over multiple days. Hemolysis assays documented PSA-dependent pore formation and lytic potential (i.e., function) of the released protoxin. MTT assays demonstrated that conditioned supernatant from PRX302-loaded, but not blank (i.e., unloaded), PLGA microparticles was highly cytotoxic to PC3 and DU145 human prostate cancer cells in the presence of exogenous PSA. Microparticle encapsulation prevented PRX302 from immediately interacting with GPI-anchored proteins as demonstrated in a competition assay, which resulted in an increased therapeutic index and significant antitumor efficacy following a single dose of PRX302-loaded microparticles in a preclinical model of prostate cancer liver metastasis with no obvious toxicity. These results document that PRX302 released from PLGA microparticles demonstrate in vivo antitumor efficacy in a clinically relevant preclinical model of metastatic prostate cancer.

PO-1821: Investigating immune microenvironment effects of radiotherapy in pre-clinical prostate cancer models

PO-1821: Investigating immune microenvironment effects of radiotherapy in pre-clinical prostate cancer models

Imaging Fibroblast Activation Protein Alpha Improves Diagnosis of Metastatic Prostate Cancer with Positron Emission Tomography.

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal, heterogeneous disease with few therapeutic strategies that significantly prolong survival. Innovative therapies for mCRPC are needed; however, the development of new therapies relies on accurate imaging to assess metastasis and monitor response. Standard imaging modalities for prostate cancer require improvement and there remains a need for selective and sensitive imaging probes that can be widely used in patients with mCRPC. We evaluated the transmembrane protease fibroblast activation protein alpha (FAP) as a targetable cell surface antigen for mCRPC. Genomic and IHC analyses were performed to investigate FAP expression in prostate cancer. Our FAP-targeted antibody imaging probe, [89Zr]Zr-B12 IgG, was evaluated by PET/CT imaging in preclinical prostate cancer models. Analysis of patient data documented FAP overexpression in metastatic disease across tumor subtypes. PET imaging with [89Zr]Zr-B12 IgG demonstrated high tumor uptake and long-term retention of the probe in the preclinical models examined. FAP-positive stroma tumor uptake of [89Zr]Zr-B12 IgG was 5-fold higher than the isotype control with mean %ID/cc of 34.13 ± 1.99 versus 6.12 ± 2.03 (n = 3/group; P = 0.0006) at 72 hours. Ex vivo biodistribution corroborated these results documenting rapid blood clearance by 24 hours and high tumor uptake of [89Zr]Zr-B12 IgG by 72 hours. Our study reveals FAP as a target for imaging the tumor microenvironment of prostate cancer. Validation of [89Zr]Zr-B12 IgG as a selective imaging probe for FAP-expressing tumors presents a new approach for noninvasive PET/CT imaging of mCRPC.

Abstract 3416: Androgen deprivation following JAK1/2 and PD-L1 inhibition improves antitumor efficacy in mouse models of Pten-deficient prostate cancer

Abstract Recently, the tumor suppressor PTEN has been shown to function as an immune modulator and its inactivation is associated with mediating tumor immune evasion. We previously examined the effects of programmed death-ligand 1 (PD-L1) immune checkpoint blockade (ICB) in transgenic mouse models of Pten-null prostate cancer. However, anti-PD-L1 monotherapy was largely ineffective in immunologically cold castration-naïve tumors from intact mice but showed some activity when administered to castrated mice. Our previous immune profiling studies also revealed that while androgen withdrawal increased tumor inflammation, it also mediated the recruitment and accumulation of immunosuppressive cells such as regulatory T cells, M2-polarized macrophages and myeloid-derived suppressor cells (MDSCs). Interleukin-6 (IL-6)/signal transducer and activator of transcription-3 (STAT3) signaling is a key immune modulating pathway that is upregulated in both intact and androgen deprived tumors. Here, we evaluate the effectiveness of targeting PD-L1 and JAK/STAT signaling in preclinical models of Pten-null prostate cancer. Intact tumor bearing conditional Pten-knockout were treated for four weeks with an a-PD-L1 blocking antibody (clone D265A, mouse/IgG1 kappa) and/or the JAK1/2 inhibitor, AZD1480. Pharmacological treatments targeting PD-L1 and JAK1/2 (PJ) combinations were also evaluated as concurrent therapy with androgen deprivation (AD) by orchidectomy (PJ+AD), and as sequential therapy; two weeks AD followed by PJ (AD&amp;gt;PJ), and one week of PT followed by AD (PJ&amp;gt;AD). Combined PD-L1/JAK followed by AD was the only treatment combination that improved antitumor immune responses over monotherapy. Notably, flow cytometry studies showed that combined PD-L1/JAK potently abrogated PD-L1 expression in circulating dendritic cells in all settings. Evidence for enhanced antitumor immune response in PJ&amp;gt;AD was supported by increased numbers circulating effector memory CD8 T cells and CD355+CD8+ T cells (CD355 Class I MHC-restricted T cell-associated molecule (CRTAM) a marker for activated CD8 T cell), and increased CD8 T cell infiltration in tumors and a reduction of CD25+CD4+ regulatory T cells. The efficacy of the PJ&amp;gt;AD treatment combination was further tested and confirmed in a subcutaneous syngeneic allograft model using mice grafted with tumor blocks from Pten-deficient castration-resistant prostate cancer tumors. Together these results indicate that pretreatment with combined PD-L1/JAK blockade can decrease the immunosuppressive effects of androgen withdrawal and have the potential to restore antitumor immune activity in Pten-deficient prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazuko Sakai, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Androgen deprivation following JAK1/2 and PD-L1 inhibition improves antitumor efficacy in mouse models of Pten-deficient prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3416.