Patient-derived Xenograft Organoid Research Articles

Immunomodulatory alpha neutrophils: The first-in-class neutrophil progenitor-based allogeneic immuno-cell therapy with cytotoxic and immunomodulatory functionality for the treatment of solid tumours.

e14523 Background: Adoptive cell therapy for the treatment of cancer has largely focused on the administration of autologous engineered αβ T cells. Although successful in some haematological malignancies, the solid tumour microenvironment (TME) poses significant challenges to the efficacy of CAR-T cells, including physical barriers, heterogeneous antigen expression or antigen escape and immunosuppressive mediators. Remodeling the immunosuppressive solid TME is pivotal to the success of immunotherapies. Recent studies have highlighted key roles for neutrophils in tumour control and in facilitating successful responses to cancer immunotherapy, with neutrophil functional heterogeneity within the TME becoming increasingly appreciated. Methods: At LIfT Biosciences we have developed the first-in-class, stem cell-derived immuno-cell therapy with anti-cancer neutrophil properties called Immunomodulatory Alpha Neutrophils (IMANs). IMANs are derived from CD34+ stem cells expanded and differentiated in proprietary media compositions in a simple and scalable GMP-compliant manufacturing process. IMANs are designed to recapitulate those neutrophil states that are both cytotoxic and capable of immunomodulating the solid TME for long-lasting tumour control. Off-the-shelf, allogeneic, innate IMANs have the potential to provide major clinical benefits in the treatment of multiple solid tumours. Results: IMANs multimodal mechanisms of action (MoA) have been demonstrated in different state-of-the-art solid tumour models, including co-culture with patient-derived xenograft organoids (PDX-O) and with 3D ex vivo patient tissues (tumour-on-a-chip). Direct antigen-independent cytotoxicity towards PDX-O from a range of solid tumour indications has been demonstrated. Additionally, and fundamental to IMANs MoA is their ability to migrate to the solid TME and recruit and activate recipients’ anti-tumour immune responses. We have demonstrated IMANs ability to rapidly migrate into the solid TME and promote recruitment of patients’ immune cells leading to enhanced tumour cell death using tumour-on-a-chip technology accompanied by daily live cell imaging. Mechanistically, co-culture with IMANs enhanced expression of co-stimulatory receptors on T and NK cells, such as OX40 and 4-1BB, as well as promoting secretion of TME-modulating factors, such as CXCL10. Furthermore, co-culture with IMANs enhanced secretion of IFN-γ by activated tumour-infiltrating lymphocytes. Conclusions: These cytotoxic and immunomodulatory functions enable IMANs to remodel the TME, leaving the solid tumour more susceptible to host immune responses.

The Transformative Role of 3D Culture Models in Triple-Negative Breast Cancer Research.

Advancements in cell culturing techniques have allowed the development of three-dimensional (3D) cell culture models sourced directly from patients' tissues and tumors, faithfully replicating the native tissue environment. These models provide a more clinically relevant platform for studying disease progression and treatment responses compared to traditional two-dimensional (2D) models. Patient-derived organoids (PDOs) and patient-derived xenograft organoids (PDXOs) emerge as innovative 3D cancer models capable of accurately mimicking the tumor's unique features, enhancing our understanding of tumor complexities, and predicting clinical outcomes. Triple-negative breast cancer (TNBC) poses significant clinical challenges due to its aggressive nature, propensity for early metastasis, and limited treatment options. TNBC PDOs and PDXOs have significantly contributed to the comprehension of TNBC, providing novel insights into its underlying mechanism and identifying potential therapeutic targets. This review explores the transformative role of various 3D cancer models in elucidating TNBC pathogenesis and guiding novel therapeutic strategies. It also provides an overview of diverse 3D cell culture models, derived from cell lines and tumors, highlighting their advantages and culturing challenges. Finally, it delves into live-cell imaging techniques, endpoint assays, and alternative cell culture media and methodologies, such as scaffold-free and scaffold-based systems, essential for advancing 3D cancer model research and development.

Abstract PO2-14-08: Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response

Abstract Organoids serve as an important preclinical model in multiple cancers including breast cancer, demonstrating preservation of biological features and feasibility of drug screening, with reasonable cost and high scalability. It has been reported that patient-derived organoids (PDOs) can be robustly derived from both primary and metastatic breast tumors, with high resemblance to source tissues in histological, genomic, and transcriptomic features, as well as consistency in drug response compared to xeno-transplantations or patients (Sachs et al, 2018). Since 2018, the Institute for Precision Medicine (IPM) has developed a panel of breast cancer organoids. The current inventory now consists of 128 PDOs from 94 primary tumors, 12 metastases, 12 rapid-autopsy tumors, and 10 normal breast tissues with a 60% success rate of culture; as well as 33 organoids from animal models including 11 patient-derived xenograft organoids (PDXOs), 15 rat (RDO) and 7 mouse (MDO) tumor organoids with 85% success rate. For PDOs, the protocol includes in-lab processing of fresh, deidentified tissue within 60 minutes of surgical operation, thanks to close collaboration with surgeons, consented patients, and an institutional biospecimen core. BC organoids have been comprehensively characterized in terms of morphology, histology, genomics, transcriptomics, and functional experiments. Key biological features are preserved. PDOs (n=56) exhibit a mutational spectrum consistent with human breast cancers. ER expression is detectable in a subset of cultures with robust estradiol response indicated by GREB1 expression; and PDOs from invasive lobular carcinoma (ILC) mostly showed more discohesive structures than PDOs from no special type (NST), with clear E-cadherin loss under immunofluorescence. We have also successfully constructed transcriptionally and genetic modified BC PDOs with shRNA and CRISPR, which showed replicable performance in proliferation assay, drug response, and signaling by western blot. Of note, longitudinal tracking of PDOs with single cell RNA-sequencing (scRNA-seq) revealed well-preserved heterogeneity compared to primary tumor, while transcriptomic clonality was mildly decreased in later passages from pilot data. Future work will include expansion of current collection from various sources, longitudinal characterization with scRNA-seq and DNA sequencing, as well as more functional studies. We aim to construct a robust resource of well-characterized and reliably-usable model bank for preclinical breast cancer research. Citation Format: Daniel Brown, Kai Ding, Fangyuan Chen, Jian Chen, Steffi Oesterreich, Peter Lucas, Priscilla McAuliffe, Jennifer Atkinson, ADRIAN LEE. Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-08.

Establishing conditions for the generation and maintenance of estrogen receptor-positive organoid models of breast cancer

Patient-derived organoid models of estrogen receptor-positive (ER+) breast cancer would provide a much-needed tool to understand drug resistance and disease progression better. However, the establishment and long-term maintenance of ER expression, function, and response in vitro remains a significant challenge. Here, we report the development of an ER+ breast tumor organoid medium (BTOM-ER) that conserves ER expression, estrogen responsiveness, and dependence, as well as sensitivity to endocrine therapy of ER+ patient-derived xenograft organoids (PDXO). Our findings demonstrate the utility of subtype-specific culture conditions that better mimic the characteristics of the breast epithelial biology and microenvironment, providing a powerful platform for investigating therapy response and disease progression of ER+ breast cancer.

Abstract 6914: Genomic landscape and stability of preclinical models in NCI's patient-derived models repository

Abstract Background: Patient-derived xenograft (PDX) models serve as a powerful tool for cancer translational research. However, it is unclear to what extent PDX models reflect the genomic intratumoral heterogeneity and genomic aberrations present within the original tumor sample. The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov/) consisting of PDX, organoids (PDOrg), and tumor cell cultures (PDC) from patients with diverse cancer histologies. We have conducted an in-depth investigation into the genomic stability of PDXs at early passaging and tumor heterogeneity in a large set of preclinical models including rare cancers. Methods: Tumor specimens were used to establish 1114 models from 1034 patients. For whole exome sequencing (WES) and RNASeq analysis, 1059 models with at least 1 PDX sample and 55 models with only a PDC or PDOrg were used. Analyzed specimens represent the original patient specimens, PDX passages P0, P1, P2, and P3+ and in vitro models. 80% of the PDX specimens were within passages P0 to P2. Results: Genomic stability in PDMR models was maintained through early passages (P0 - P2) and across independent lineages in PDMR models based on the following observations: 1) variant allele frequencies (VAF) of somatic driver mutations showed no major deviations (exceeding ±10%) when using the stromal fraction corrected VAF from originator data over PDX passages; 2) the median change in the fraction of genome impacted by copy number alterations (CNA) from the originator specimens through passages P0 - P2 was statistically inconsequential, although there was a significant increase in CNA fraction, from 5.3% at P0 initially to 18% by P3; 3) gene expression profiles of specimens within a given PDX model formed clusters in principal component analysis (PCA) plots and showed high correlation (>99.5% models with a Spearman coefficient of >= 0.8). The majority of the PDC and PDOrg specimens exhibited similar findings when compared to the original tumor samples. Furthermore, the positive percent agreement (PPA) was performed to measure the similarity between the patient's tumor and all available derived PDX specimens, with a median PPA over 90%, indicating a high level of retained heterogeneity within the PDX models. Lastly, 69% of models in NCI PDMR had predictive biomarkers, ranging from OncoKB level of evidence 1-4, which could be used to evaluate targeted therapeutics in preclinical studies. Conclusion: The NCI PDMR has a large and histologically diverse cancer representation. In this analysis, the PDXs exhibited genomic stability within early passages and preserved the majority of tumor heterogeneity observed in the patient specimens. NCI PDMR thus represents a valuable resource for researchers interested in preclinical drug screening or other investigations. Citation Format: Ting-Chia Chang, Biswajit Das, Li Chen, Peter I-Fan Wu, Yvonne A. Evrard, Rini Pauly, Dianne L. Newton, Shahanawaz Jiwani, Sergio Y. Alcoser, Luke H. Stockwin, Corinne E. Camalier, Tomas Forbes, Nikitha Nair, Alyssa Chapman, Lindsay Dutko, Michael Mullendore, Tara Grinnage-Pulley, Kimberly Klarmann, Sarah B. Miller, Chris A. Karlovich, Alice P. Chen, P Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. Genomic landscape and stability of preclinical models in NCI's patient-derived models repository [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 6914.

Abstract 4727: Large scale drug response comparison across different neuroblastoma cell model systems

Abstract Traditionally neuroblastoma research has predominantly used 2D cell lines, which, despite their simplicity, often fall short in mirroring the physiological complexity of actual tumors. Recently, 3D models like patient-derived tumor organoids (PDO) and patient-derived xenografts (PDX) have emerged as superior alternatives, offering more realistic predictions of patient treatment responses. This study aims to discern the differences in drug response between conventional 2D cell lines and the more advanced 3D neuroblastoma models - specifically, patient-derived organoids (nPDO) and patient-derived xenograft organoids (nPDXO). Employing high-throughput drug screening of 528 cancer drugs, we evaluated the drug responses across traditional 2D cell lines, nPDOs, and nPDXOs. Additionally, focused experiments with 29 selected drugs were conducted to determine the influence of culture topology on drug efficacy. The findings were compared to a study by Hansson et al.1 that used nPDXO. Our study discovered a pronounced difference in drug sensitivity between 2D cell lines and 3D models, with 3D models showing heightened sensitivity. Interestingly, 2D cell cultures had a more heterogenous drug response compared to 3D models. The drug response correlation between nPDOs and nPDXOs was relatively high (Spearman ~0.8), indicating consistent responses across these models. Our comparison underscores the impact of model system choice on drug response studies in neuroblastoma. The study highlights important differences in drug response between neuroblastoma 2D and 3D cell culture models. However, only subtle differences were found between PDO and PDX-derived organoid lines, despite differences in treatment centers and media compositions. This suggests that while the choice of cell culture topology greatly impacts drug response, PDO and PDX derived lines can largely be used interchangeably for drug screening experiments.

Abstract 4596: Targeting arginine methylome in 9p21/MTAP-deleted malignant cancers with a next generation PRMT5-specific inhibitor CTS3497

Abstract It remains largely challenging to treat cancer patients with chromosome 9p21-deletion which occurs in approximately 15% of human cancers. Recently, specific targeting of protein arginine methyltransferase 5 (PRMT5), a key member of the type-II PRMT family and a master epigenetic modulator of arginine methylome essential for cancer progression, has emerged as a promising therapy in various hematological and solid tumors with MTAP deletion serving as a precision biomarker. To optimize the potential of targeting PRMT5, we developed CTS3497, a potent, brain-penetrable, orally bioavailable MTA-cooperative PRMT5 inhibitor, using our EpigenPLUSTM platform. The overall excellent drug-like properties of CTS3497 support fast track IND application, and it is anticipated to enter Phase 1 trials in 2024. CTS3497 demonstrated potent PRMT5 inhibitory activity in cellular pharmacodynamics (PD) assays, evidenced by the suppression of symmetric dimethylarginine (SDMA). Additionally, it exhibited profound cell growth inhibition with a low single-digit nM IC50 and 171-fold high selectivity for MTAPnull over isogenic MTAPwt cell lines. Moreover, CTS3497 displayed strong inhibitory effects on cell growth across diverse panels of MTAPnull cancer cell lines and patient-derived xenograft organoids (PDXOs) spanning various lineages. In vivo, oral administration of CTS3497 resulted in significant tumor regression in multiple MTAP-deleted xenograft models, notably MTAPnull brain tumors. The observed anti-tumor activity correlated with PRMT5 modulation, as indicated by reduced SDMA levels. Impressively, sustained tumor growth inhibition led to the complete tumor regression, with no regrowth even after discontinuation of CTS3497 treatment. The brain penetration property of CTS3497 underscores its therapeutic potential for both primary brain tumors, and brain metastases arising from specific cancers like lung cancer. At the molecular level, PRMT5 inhibition by CTS3497 induced substantial and distinctive RNA splicing defects and differentially expressed gene signatures, impacting key cellular activities downstream of arginine and epigenetic reprogramming in MTAPnull cancer cells. Furthermore, a synergistic anti-tumor effect was observed in multiple MTAP-deleted tumors when combining PRMT5 inhibition with CTS2190, an investigational type-I PRMT inhibitor currently in phase I/II clinical trials. In summary, CTS3497, a PRMT5 inhibitor selectively targeting MTAPnull tumor cells, demonstrated robust and durable anti-tumor responses both in vitro and in vivo across diverse lineages of MTAP-deleted tumors. Its superior ADME properties and favorable safety profiles observed in preclinical studies mark a breakthrough in the next generation epigenetic therapy. CTS3497 emerges as a promising therapeutic option for patients with MTAP-deleted malignancies. Citation Format: Yilin Liu, Long Wang, Hui Shi, Yuanyuan Liu, Qiugeng Ouyang, Xingnian Fu, Meng Wang, Jiannan Guo, Yiqin Wang, Youzhen Wang, Guoliang Xu, Yuan Mi, Haiping Wu. Targeting arginine methylome in 9p21/MTAP-deleted malignant cancers with a next generation PRMT5-specific inhibitor CTS3497 [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 4596.

Abstract 4567: Dual targeting of HSP70 and AURKA improves treatment in neuroendocrine prostate cancer

Abstract Background: Neuroendocrine prostate cancer (NEPC) is the most aggressive type of prostate cancer with no effective treatments. Therefore, there is an urgent need to develop new treatment strategies. N-Myc plays a key role in driving NEPC progression, and Aurora kinase A (AURKA) prevents N-Myc degradation by interact with N-Myc and disallowing the intervention of ubiquitin ligases. The AURKA inhibitor Alisertib inhibits NEPC tumor growth by disrupting N-Myc signaling, but it is failed in a phase II clinical trial. In this study, we tested the synergy of novel HSP70 allosteric inhibitor JG231 and Alisertib in NEPC cells, organoids, and patient derived xenograft (PDX) models, which will provide new therapeutic strategies for the NEPC treatment. Methods: RT-PCR, western blotting, or immunohistochemistry (IHC) were used to determine the expression of N-Myc, neuroendocrine maker (NSE, SYP, CHGA, etc) in different prostate cancer cell lines and PDX tumors. After the knockdown of HSP70 and AURKA by siRNA transfection, cell proliferation and N-Myc expression were assessed by using cell viability assay, RT-PCR and Western blotting respectively. The co-immunoprecipitation assay was performed to detect the N-Myc ubiquitination. RNA-sequencing, gene set enrichment analysis was employed to determine the changes of JG231 on gene programs. The effects of JG231 and Alisertib on the proliferation were examined in NEPC cell lines and PDX organoids models. Results: We determined that PC3, CWR22Rv1, H660 cell lines, LuCaP49, and LuCaP93 PDX tumors had significantly increased the expression of N-Myc and neuroendocrine makers. Knockdown of HSP70 or using the HSP70 inhibitor, JG231, significantly inhibit the growth of H660 and CWR22Rv1 cells and leads to a synergistic effect with Alisertib (p<0.001). JG231 combined with Alisertib also inhibits growth and induces the death of LuCaP93 and H660 tumor derived organoids in a dose-dependent manner (p<0.001). Mechanistically, JG231 inhibits the protein expression of N-Myc through the ubiquitin-proteasome system and promote STUB1 to enter the nucleus to bind to N-Myc. Conclusions: JG231, a novel HSP70 inhibitor, can improves the treatment efficacy of AURKA inhibitor Alisertib against NEPC through the regulation of N-Myc protein homeostasis. Citation Format: Pengfei Xu, Joy C. Yang, Bo Chen, Shu Ning, Leyi Wang, Christopher Nip, Christopher P. Evans, Marc A. Dall'Era, Allen C. Gao, Jason Gestwicki, Chengfei Liu. Dual targeting of HSP70 and AURKA improves treatment in neuroendocrine 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 4567.

DHODH inhibition represents a therapeutic strategy and improves abiraterone treatment in castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is an aggressive disease with poor prognosis, and there is an urgent need for more effective therapeutic targets to address this challenge. Here, we showed that dihydroorotate dehydrogenase (DHODH), an enzyme crucial in the pyrimidine biosynthesis pathway, is a promising therapeutic target for CRPC. The transcript levels of DHODH were significantly elevated in prostate tumors and were negatively correlated with the prognosis of patients with prostate cancer. DHODH inhibition effectively suppressed CRPC progression by blocking cell cycle progression and inducing apoptosis. Notably, treatment with DHODH inhibitor BAY2402234 activated androgen biosynthesis signaling in CRPC cells. However, the combination treatment with BAY2402234 and abiraterone decreased intratumoral testosterone levels and induced apoptosis, which inhibited the growth of CWR22Rv1 xenograft tumors and patient-derived xenograft organoids. Taken together, these results establish DHODH as a key player in CRPC and as a potential therapeutic target for advanced prostate cancer.

Patient- and xenograft-derived organoids recapitulate pediatric brain tumor features and patient treatments.

Brain tumors are the leading cause of cancer-related death in children. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of pediatric brain cancers are limited and hard to establish. We present a protocol that enables efficient generation, expansion, and biobanking of pediatric brain cancer organoids. Utilizing our protocol, we have established patient-derived organoids (PDOs) from ependymomas, medulloblastomas, low-grade glial tumors, and patient-derived xenograft organoids (PDXOs) from medulloblastoma xenografts. PDOs and PDXOs recapitulate histological features, DNA methylation profiles, and intratumor heterogeneity of the tumors from which they were derived. We also showed that PDOs can be xenografted. Most interestingly, when subjected to the same routinely applied therapeutic regimens, PDOs respond similarly to the patients. Taken together, our study highlights the potential of PDOs and PDXOs for research and translational applications for personalized medicine.

MAP kinase ERK5 modulates cancer cell sensitivity to extrinsic apoptosis induced by death-receptor agonists

Death receptor ligand TRAIL is a promising cancer therapy due to its ability to selectively trigger extrinsic apoptosis in cancer cells. However, TRAIL–based therapies in humans have shown limitations, mainly due inherent or acquired resistance of tumor cells. To address this issue, current efforts are focussed on dissecting the intracellular signaling pathways involved in resistance to TRAIL, to identify strategies that sensitize cancer cells to TRAIL-induced cytotoxicity. In this work, we describe the oncogenic MEK5-ERK5 pathway as a critical regulator of cancer cell resistance to the apoptosis induced by death receptor ligands. Using 2D and 3D cell cultures and transcriptomic analyses, we show that ERK5 controls the proteostasis of TP53INP2, a protein necessary for full activation of caspase-8 in response to TNFα, FasL or TRAIL. Mechanistically, ERK5 phosphorylates and induces ubiquitylation and proteasomal degradation of TP53INP2, resulting in cancer cell resistance to TRAIL. Concordantly, ERK5 inhibition or genetic deletion, by stabilizing TP53INP2, sensitizes cancer cells to the apoptosis induced by recombinant TRAIL and TRAIL/FasL expressed by Natural Killer cells. The MEK5-ERK5 pathway regulates cancer cell proliferation and survival, and ERK5 inhibitors have shown anticancer activity in preclinical models of solid tumors. Using endometrial cancer patient-derived xenograft organoids, we propose ERK5 inhibition as an effective strategy to sensitize cancer cells to TRAIL-based therapies.

P04.08.A PATIENT-DERIVED ORGANOIDS FOR MODELING AND CHARACTERIZING PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Central nervous system (CNS) tumors are the most common solid tumors in the pediatric population and the leading cause of death among childhood cancers. They present common features related to the development and produce symptoms based on their location, rate of tumor growth and age of the child. Tumors of embryonal origin, such as medulloblastoma or primitive neuroectodermal tumor, have a higher incidence in younger patients, whereas older ones tend to present tumors of glial origin. Most of CNS-embryonal tumors are highly malignant and consequently leading to poor prognoses. Despite the understanding of their underlying molecular landscape that has helped in clinical advances to improve life expectancies of the patients, there is still an emerging need to minimize morbidity and manage the long-term therapy effects. MATERIAL AND METHODS Human samples were collected according to protocols approved by the institutional review board with written consent. Patient-Derived Organoids (PDOs) and Patient-Derived Xenograft Organoids (PDXOs) were obtained using scalpels by chopping in small pieces the tumor tissue and were grown in PDOs medium. PDOs and PDXOs were analyzed through DNA methylation, immunostainings, sc-RNA sequencing and orthotopic nude mice engraftment. Therapeutic regimens were adapted according to patients’ treatment protocols depending on the tumor of origin. RESULTS Developing new models to better understand the complexity and heterogeneity of pediatric tumors and to test personalized treatment strategies remains a clinical challenge. We tried to answer this medical need with PDOs and PDXOs established through the direct in vitro culture of different kinds of primary tumors (ependymoma EPN, medulloblastoma MB and low-grade glial tumors) and MB patient derived xenograft (PDX)-derived tumors. We have developed a method that recapitulates mutational profiles, histological features, and cellular heterogeneity of the corresponding parental tumors. We have obtained promising results from the DNA methylation profile analysis, nowadays used for an accurate routine CNS tumors diagnosis. Through this new model, we confirmed the action of clinically used therapeutic regimens, i.e., for EPN with ZFTA-RELA fusion and G3 MB-derived PDOs. PDOs were subjected to the same treatment the patients underwent and responded similarly to the patients. CONCLUSION We found a suitable in vitro culture condition to generate and maintain PDOs from different kinds of pediatric brain tumors. Their reliability has been proved by different analyses, confirming they still resemble the parental tumors. Through this new model, we confirmed the action of clinically used therapeutic regimens, suggesting its potential at the translation level as a reliable tool for testing personalized medicine, drug screening and development of newer and more specific therapeutic strategies.

Targeting Mcl-1 by a small molecule NSC260594 for triple-negative breast cancer therapy

Triple-negative breast cancers (TNBCs) are aggressive forms of breast cancer and tend to grow and spread more quickly than most other types of breast cancer. TNBCs can neither be targeted by hormonal therapies nor the antibody trastuzumab that targets the HER2 protein. There are urgent unmet medical needs to develop targeted drugs for TNBCs. We identified a small molecule NSC260594 from the NCI diversity set IV compound library. NSC260594 exhibited dramatic cytotoxicity in multiple TNBCs in a dose-and time-dependent manner. NSC260594 inhibited the Myeloid cell leukemia-1 (Mcl-1) expression through downregulation of Wnt signaling proteins. Consistent with this, NSC260594 treatment increased apoptosis, which was confirmed by using an Annexin-V/PI assay. Interestingly, NSC260594 treatment reduced the cancer stem cell (CSC) population in TNBCs. To make NSC260594 more clinically relevant, we treated NSC260594 with TNBC cell derived xenograft (CDX) mouse model, and with patient-derived xenograft (PDX) organoids. NSC260594 significantly suppressed MDA-MB-231 tumor growth in vivo, and furthermore, the combination treatment of NSC260594 and everolimus acted synergistically to decrease growth of TNBC PDX organoids. Together, we found that NSC260594 might serve as a lead compound for triple-negative breast cancer therapy through targeting Mcl-1.

Abstract B020: BCL2 expression is enriched in AR-independent advanced prostate cancer

Abstract Background The widespread use of highly potent androgen receptor (AR) inhibitors has led to an increasing emergence of AR-independent castration-resistant prostate cancer (CRPC). AR independence, typically driven by loss of AR expression and/or trans-differentiation to a neuroendocrine or basal phenotype, lacks effective therapeutic strategies. The anti-apoptotic protein BCL2 is upregulated in neuroendocrine tumours and has been proposed as a therapeutic target for this subset of advanced prostate cancers. Objective To characterise AR expression in advanced PC, to evaluate its association with BCL2, to elucidate their clinical significance, and to explore whether BCL2 may represent a valid therapeutic target and/or biomarker. Methods AR and BCL2 expression were immunohistochemically scored in two independent CRPC cohorts (431 biopsies from 247 patients), including 62 matched same-patient castration-sensitive PC (CSPC) biopsies. Scores were associated with retrospectively collected clinical data including overall survival (OS) and response to AR-targeting therapy. BCL2 protein expression was also determined in 28 primary prostate samples (19 patients). Transcriptome data from two independent CRPC cohorts were analysed, including for associations between BCL2 expression and gene expression signatures. A patient-derived xenograft-organoid (PDX-O) CRPC model with high expression of BCL2 was treated with multiple BH3 mimetics. Results and limitations We demonstrate that AR protein loss emerges with castration resistance and occurs in subset of metastatic CRPC (mCRPC) (5%) associating with worse OS (51 vs 18 months from CRPC diagnosis, hazard ratio [HR] 5.52 [2.83-10.78], p<0.001). BCL2 protein expression is enriched in AR-negative mCRPC (p<0.001), associating with worse OS (53 vs 19 months from CRPC diagnosis, HR 2.80 [1.40-5.57], p=0.002) and resistance to AR-targeting therapy (≥50% PSA response: 48% vs 13%), irrespective of AR expression status. BCL2 mRNA expression associates with several key signalling pathways known to promote stemness and lineage plasticity, including EMT and IL6/JAK/STAT3. Furthermore, BCL2 is enriched in basal prostate cells, whose expression profile has been linked with aggressive subtypes of mCRPC, including small cell neuroendocrine CPRC. We show that a high BCL2-expressing PDX-O is resistant to BCL2 inhibition, with functional redundancy between the anti-apoptotic BCL2 family proteins. BCLXL and MCL1 are highly expressed in BCL2-positive mCRPC and may drive resistance to BCL2 targeting. Utilising a post-mortem mCRPC biopsy cohort, we show intra-patient heterogeneity in BCL2 expression. These findings highlight the need for combination therapies. Further studies are required to dissect the interaction between AR and BCL2, and to probe the functional role of BCL2 in this subset of mCRPC. Conclusions BCL2-positivity associates with AR-independence, worse OS, and resistance to AR-targeting in CRPC. Combination strategies are required to optimise response to BCL2 inhibition and to combat heterogeneity. Citation Format: Daniel Westaby, Juan M. Jimenez-Vacas, Ines Figueiredo, Claire Pettinger, Bora Gurel, Denisa Bogdan, Jan Rekowski, Lorenzo Buroni, Antje Neeb, Ruth Riisnaes, Mateus Crespo, Susana Miranda, Ana Ferreira, Daniel Nava Rodrigues, George Seed, Claudia Bertan, Maria de los Dolores Fenor de la Maza, Christina Guo, Juliet Carmichael, Rafael Grochot, Khobe Chandran, Andreas Varkaris, Steven P. Balk, Wei Yuan, Suzanne Carreira, Peter S. Nelson, Michael Haffner, Eva Corey, Johann de Bono, Adam Sharp. BCL2 expression is enriched in AR-independent advanced 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 B020.

MP20-16 NOVEL FUNCTION OF PTUPB IN AKR1C3/AR/AR-V7 AXIS INHIBITION AND ENZALUTAMIDE SENSITIZATION IN ADVANCED PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023MP20-16 NOVEL FUNCTION OF PTUPB IN AKR1C3/AR/AR-V7 AXIS INHIBITION AND ENZALUTAMIDE SENSITIZATION IN ADVANCED PROSTATE CANCER Joy Yang, Pengfei Xu, Bo Chen, Shu Ning, Hans Adomat, Sung-Hee Hwang, Allen Gao, Martin Gleave, Bruce Hammock, Christopher Evans, and Chengfei Liu Joy YangJoy Yang More articles by this author , Pengfei XuPengfei Xu More articles by this author , Bo ChenBo Chen More articles by this author , Shu NingShu Ning More articles by this author , Hans AdomatHans Adomat More articles by this author , Sung-Hee HwangSung-Hee Hwang More articles by this author , Allen GaoAllen Gao More articles by this author , Martin GleaveMartin Gleave More articles by this author , Bruce HammockBruce Hammock More articles by this author , Christopher EvansChristopher Evans More articles by this author , and Chengfei LiuChengfei Liu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The steroidogenic enzyme Aldo-keto Reductase 1C3 (AKR1C3) is a major factor that contributes to the progression of castration-resistant prostate cancer (CRPC) and treatment failure. However, none of the available AKR1C3 inhibitors has shown promising results in clinical settings. Thus, there is an unmet need to develop superior AKR1C3 inhibitors for clinical testing. METHODS: PTUPB was synthesized by linking two pharmacophores, celecoxib and TPPU. In vitro and in vivo pharmacological activities of PTUPB in combination with enzalutamide were evaluated in several enzalutamide-resistant cell lines, conditional reprogrammed cell cultures, and patient-derived organoid models. The mechanism of action of PTUPB was examined via RNA sequence analysis, immunoblot analysis, co-immunoprecipitation, steroid measurement, and enzyme activity assays. Animal studies using PTUPB in combination with enzalutamide were performed in the castration-relapse VCaP tumor model. RESULTS: AKR1C3 levels are reciprocally regulated by the full-length androgen receptor (AR-FL) by binding to the distal enhancer region of AKR1C3. A novel function of PTUPB in AKR1C3 inhibition was discovered: at the same dose (0.5 µM) PTUPB suppressed the AKR1C3 activity more than indomethacin (60% vs. 40%) and celeboxib (60% vs. 0, p<0.05). And at the same dose (20 µM), PTUPB was more effective than indomethacin (98% vs. 22% of inhibition, p<0.05) and celecoxib (98% vs. 60% inhibition, p<0.05) in inhibiting CRPC cell growth. PTUPB synergizes with enzalutamide treatment for tumor suppression and gene signature regulation. Combination treatment with PTUPB and enzalutamide provides benefits by blocking AR and its variant (AR-V7) signaling, which inhibits the growth of VCaP castration-relapse tumors with 2.8-fold more inhibition (p<0.05) in tumor weights between the single (PTUPB) and combination treatments (Enza+PTUPB). There was also more inhibition in the growth of the patient-derived xenograft organoids by PTUPB alone or in combination with Enza. CONCLUSIONS: Targeting the ARK1C3/AR/AR-V7 axis with PTUPB and enzalutamide may overcome drug resistance to AR signaling inhibitors in advanced prostate cancer. 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: e281 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joy Yang More articles by this author Pengfei Xu More articles by this author Bo Chen More articles by this author Shu Ning More articles by this author Hans Adomat More articles by this author Sung-Hee Hwang More articles by this author Allen Gao More articles by this author Martin Gleave More articles by this author Bruce Hammock More articles by this author Christopher Evans More articles by this author Chengfei Liu More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract P3-08-10: Combining multiomics and histological assessment to identify patient derived xenograft models of invasive lobular carcinoma

Abstract Background Most breast cancers (~85%) are of no special histologic subtype (NST), and the most common special subtype is invasive lobular cancer (ILC). ILC accounts for 10-15% of all breast cancers, and there will be ~40,000 new cases in 2022 in the US alone. If considered an “independent” cancer type, ILC is the 6th most common cancer in women. The pathognomonic feature of ILC is loss of E-cadherin (CDH1). The resulting lack of adherens junctions causes the unique single-file growth pattern of discohesive ILC cells, which decreases the ability for detection by mammography, in turn resulting in late detection and hence larger tumors. Although ILCs show better prognostic factors than NST, patients with ILC have worse long-term outcome, which is not well understood. Additionally, ILC has historically been understudied, which is in part due to lack of appropriate research models. For example, the Cancer Cell Line Encyclopedia (CCLE) contains 54 NST cell lines but only 2 ILC cell lines, and only a limited number of patient-derived xenograft (PDX) models are evident in the published literature. There is a critical need for additional in vitro and in vivo models to study ILC biology, as well as to test targeted therapies. ILC PDX and patient derived xenograft organoids (PDXO) are particularly valuable tools to enable target validation and assess drug treatment response. Methods To identify and validate new ILC PDX models, we used Champions Oncology’s Lumin Bioinformatics to screen Champions’ collection of breast cancer models (n=126) for PDX harboring CDH1 mutation and/or low E-cadherin expression. We performed histological analysis on selected PDX models including H&amp;E staining, and immunohistochemical assessment of E-cadherin, P120, estrogen receptor, progesterone receptor, HER2 and Ki67. Models with 2+ HER2 staining were assessed by FISH. All staining was interpreted by a certified breast pathologist. PDX tumor tissue was further used to develop PDXO models. Results Using Champions Oncology’s Lumin tool, we identified 10 putative ILC PDX models based upon CDH1 mutation, low E-cadherin mRNA expression, or clinical annotation of ILC (Table 1). Of the 10 PDX models analyzed, two cases were clinically annotated as ILC, while the remainder were classified as ‘carcinoma’ (n=5) or as NST (n=3). Histologic analysis revealed loss of E-cadherin and cytoplasmic P120 (lobular pattern) in 8/10 models assessed, and pathologic assessment confirmed these as having a lobular histology. IHC analysis classified these PDXs as 5 TNBC and 5 ER+ tumors, with none showing amplification of HER2 by FISH. All tumors demonstrated high (35%, n=1) or very high (&amp;gt;55%, n=9) Ki67 proliferation marker levels. We further developed PDXOs from one PDX model as proof of concept, and the resulting organoid demonstrated classic ‘grape-like’ ILC morphology. Conclusion Our study demonstrates how existing PDX banks with in-depth multi-omic and pathology analyses can be interrogated to identify models of unique histological and molecular subtypes of breast cancer. Of the PDX models selected from Champions Oncology’s breast cancer cohort, 7 models were classified as ILC either through re-classification from NST/carcinoma to ILC or confirmation of ILC histology. In addition, 1 PDX was re-classified as mixed type. Some of these models are ER+/HER- and thus have the classic molecular features of ILC. Our collaborative omics guided approach allows for reclassification of PDX models to increase available research models for unique breast cancer subtypes such as ILC which in turn will enhance translational research in unique histological subtypes of breast cancer. Citation Format: Jennifer M. Atkinson, Megan Yates, Daniel D. Brown, Jagmohan Hooda, Rohit Bhargava, Paolo Schiavini, Marianna Zipeto, Steffi Oesterreich, ADRIAN V. LEE. Combining multiomics and histological assessment to identify patient derived xenograft models of invasive lobular carcinoma [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-10.

Novel inhibition of AKR1C3 and androgen receptor axis by PTUPB synergizes enzalutamide treatment in advanced prostate cancer.

Castration-resistant prostate cancer (CRPC) is the main driving force of mortality in prostate cancer patients. Among the parameters contributing to the progression of CRPC and treatment failure, elevation of the steroidogenic enzyme AKR1C3 and androgen receptor variant 7 (AR-V7) are frequently reported. The AKR1C3/AR-V7 complex has been recognized as a major driver for drug resistance in advanced prostate cancer. Herein we report that the level of AKR1C3 is reciprocally regulated by the full-length androgen receptor (AR-FL) through binding to the distal enhancer region of the AKR1C3 gene. A novel function of PTUPB in AKR1C3 inhibition was discovered and PTUPB showed more effectiveness than indomethacin and celecoxib in suppressing AKR1C3 activity and CRPC cell growth. PTUPB synergizes with enzalutamide treatment in tumor suppression and gene signature regulation. Combination treatments with PTUPB and enzalutamide provide benefits by blocking AR/AR-V7 signaling, which inhibits the growth of castration relapsed VCaP xenograft tumors and patient-derived xenograft organoids. Targeting of the ARK1C3/AR/AR-V7 axis with PTUPB and enzalutamide may overcome drug resistance to AR signaling inhibitors in advanced prostate cancer.

Splice-switch oligonucleotide-based combinatorial platform prioritizes synthetic lethal targets CHK1 and BRD4 against MYC-driven hepatocellular carcinoma.

Deregulation of MYC is among the most frequent oncogenic drivers in hepatocellular carcinoma (HCC). Unfortunately, the clinical success of MYC-targeted therapies is limited. Synthetic lethality offers an alternative therapeutic strategy by leveraging on vulnerabilities in tumors with MYC deregulation. While several synthetic lethal targets of MYC have been identified in HCC, the need to prioritize targets with the greatest therapeutic potential has been unmet. Here, we demonstrate that by pairing splice-switch oligonucleotide (SSO) technologies with our phenotypic-analytical hybrid multidrug interrogation platform, quadratic phenotypic optimization platform (QPOP), we can disrupt the functional expression of these targets in specific combinatorial tests to rapidly determine target-target interactions and rank synthetic lethality targets. Our SSO-QPOP analyses revealed that simultaneous attenuation of CHK1 and BRD4 function is an effective combination specific in MYC-deregulated HCC, successfully suppressing HCC progression in vitro. Pharmacological inhibitors of CHK1 and BRD4 further demonstrated its translational value by exhibiting synergistic interactions in patient-derived xenograft organoid models of HCC harboring high levels of MYC deregulation. Collectively, our work demonstrates the capacity of SSO-QPOP as a target prioritization tool in the drug development pipeline, as well as the therapeutic potential of CHK1 and BRD4 in MYC-driven HCC.

Endocrine Therapy-Resistant Breast Cancer Cells Are More Sensitive to Ceramide Kinase Inhibition and Elevated Ceramide Levels Than Therapy-Sensitive Breast Cancer Cells.

Simple SummaryEndocrine therapy (ET) resistance is a major problem in estrogen receptor-positive breast cancer patients. Since there have been few lipidomic studies in ET resistance and sphingolipids are heavily implicated in multidrug-resistant and chemotherapy-resistant cancers, we aimed to investigate the sphingolipidome of tamoxifen-resistant breast cancer cells in search of a unique sphingolipid profile that can potentially be exploited therapeutically. We found that ET-resistant breast cancer cells maintain a lower level of ceramides for their survival. In order to achieve this, they are dependent on ceramide kinase (CERK), the activity of which helps maintain low endogenous ceramide levels, therefore promoting tamoxifen-resistant cell survival. Targeting CERK can therefore represent an opportunity to target therapy-resistant breast tumors and improve the patient outcome for women with ET-resistant disease.ET resistance is a critical problem for estrogen receptor-positive (ER+) breast cancer. In this study, we have investigated how alterations in sphingolipids promote cell survival in ET-resistant breast cancer. We have performed LC-MS-based targeted sphingolipidomics of tamoxifen-sensitive and -resistant MCF-7 breast cancer cell lines. Follow-up studies included treatments of cell lines and patient-derived xenograft organoids (PDxO) with small molecule inhibitors; cytometric analyses to measure cell death, proliferation, and apoptosis; siRNA-mediated knockdown; RT-qPCR and Western blot for gene and protein expression; targeted lipid analysis; and lipid addback experiments. We found that tamoxifen-resistant cells have lower levels of ceramides and hexosylceramides compared to their tamoxifen-sensitive counterpart. Upon perturbing the sphingolipid pathway with small molecule inhibitors of key enzymes, we identified that CERK is essential for tamoxifen-resistant breast cancer cell survival, as well as a fulvestrant-resistant PDxO. CERK inhibition induces ceramide-mediated cell death in tamoxifen-resistant cells. Ceramide-1-phosphate (C1P) partially reverses CERK inhibition-induced cell death in tamoxifen-resistant cells, likely through lowering endogenous ceramide levels. Our findings suggest that ET-resistant breast cancer cells maintain lower ceramide levels as an essential pro-survival mechanism. Consequently, ET-resistant breast cancer models have a unique dependence on CERK as its activity can inhibit de novo ceramide production.

PD07-04 PRE-CLINICAL STUDIES FOR ADVANCING CIRMTUZUMAB-BASED ANTI-ROR1 THERAPIES IN METASTATIC PROSTATE CANCER

You have accessJournal of UrologyCME1 May 2022PD07-04 PRE-CLINICAL STUDIES FOR ADVANCING CIRMTUZUMAB-BASED ANTI-ROR1 THERAPIES IN METASTATIC PROSTATE CANCER Michelle Muldong, Sanghee Lee, Theresa Mendoza, Danielle Burner, Christina Wu, Jamillah Murtadha, Evodie Koutouan, Naomi Pineda, Gabriel Pineda, Kathleen Lennon, Hao Pham, Karl Willert, Nicholas Cacalano, Catriona Jamieson, Terry Gaasterland, Christopher Kane, Anna Kulidjian, and Christina Jamieson Michelle MuldongMichelle Muldong More articles by this author , Sanghee LeeSanghee Lee More articles by this author , Theresa MendozaTheresa Mendoza More articles by this author , Danielle BurnerDanielle Burner More articles by this author , Christina WuChristina Wu More articles by this author , Jamillah MurtadhaJamillah Murtadha More articles by this author , Evodie KoutouanEvodie Koutouan More articles by this author , Naomi PinedaNaomi Pineda More articles by this author , Gabriel PinedaGabriel Pineda More articles by this author , Kathleen LennonKathleen Lennon More articles by this author , Hao PhamHao Pham More articles by this author , Karl WillertKarl Willert More articles by this author , Nicholas CacalanoNicholas Cacalano More articles by this author , Catriona JamiesonCatriona Jamieson More articles by this author , Terry GaasterlandTerry Gaasterland More articles by this author , Christopher KaneChristopher Kane More articles by this author , Anna KulidjianAnna Kulidjian More articles by this author , and Christina JamiesonChristina Jamieson More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002526.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Increased levels of the Wnt ligand, WNT5A, are correlated with bone metastatic, castrate resistant PCa (CRPC). Wnt signaling is comprised of canonical (β-catenin dependent) and noncanonical pathways which are critical for normal development and physiology. Alterations are frequently associated with tumorigenesis and metastasis in many cancers including prostate cancer. Noncanonical Wnt5A signaling is mediated in part through ROR1, a fetal oncoprotein for which a therapeutic ROR1 inhibitory antibody, Cirmtuzumab, has been developed. Its safety has been clinically proven in trials for chronic lymphocytic leukemia (CLL) and metastatic breast cancer.Hypothesis: WNT5A may activate a stem-cell-like program via ROR1 that leads to castration resistance in bone metastatic prostate cancer. The anti-ROR1 biologic, Cirmtuzumab, may inhibit this mechanism of CRPC and re-sensitize bone metastatic prostate cancer to standard of care therapies. METHODS: We used patient-derived xenograft (PDX) bone metastatic prostate cancer models and PCa cell lines to test mechanism of action of a new therapeutic target, the WNT5A/ROR1 signaling pathway in prostate cancer for which a therapeutic ROR1 inhibitor antibody, Cirmtuzumab, has been developed and clinically tested in CLL and metastatic breast cancer patients. RESULTS: Preclinical studies using RNASeq, qRT-PCR, FACS and Westerns showed increased expression of ROR1 in the neuroendocrine prostate cancer cell lines, PC3 and DU145, and a patient derived xenograft (PDX) model of small cell bone metastatic prostate cancer, PCSD13. Pretreatment of the PCSD13 PDX with Cirmtuzumab increased the efficacy of the taxane docetaxel at inhibiting tumor growth. Intravenous infusion of anti-ROR1 CAR-T cells into mice bearing subcutaneous, ROR1-expressing, PC3 xenografts, showed marked reduction in tumor growth. ROR1 Knock out using CRISPR-Cas9 in PC3 and DU145 cells showed specificity of Cirmtuzumab-based immunotherapies. We have developed immunohistochemistry (IHC) assays to detect ROR1 in patient biopsy samples which will allow us to define the prostate cancer patient population that expressed ROR1 and to be able to evaluate the treatment in patients for a future clinical trial. CONCLUSIONS: ROR1 was expressed at high levels on castration resistant small cell PCa and neuroendocrine PCa models in PCa cell lines and PDX models. Cirmtuzumab has demonstrated efficacy in our patient derived xenograft organoid cultures. These studies have formed the rationale for development of a Phase 1b clinical trial of Cirmtuzumab in combination with docetaxel in metastatic CRPC patients. Source of Funding: Dept of Defense CDMRP PCRP Impact Award, JM Foundation © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e99 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michelle Muldong More articles by this author Sanghee Lee More articles by this author Theresa Mendoza More articles by this author Danielle Burner More articles by this author Christina Wu More articles by this author Jamillah Murtadha More articles by this author Evodie Koutouan More articles by this author Naomi Pineda More articles by this author Gabriel Pineda More articles by this author Kathleen Lennon More articles by this author Hao Pham More articles by this author Karl Willert More articles by this author Nicholas Cacalano More articles by this author Catriona Jamieson More articles by this author Terry Gaasterland More articles by this author Christopher Kane More articles by this author Anna Kulidjian More articles by this author Christina Jamieson More articles by this author Expand All Advertisement PDF DownloadLoading ...