Patient-derived Organoid Models Research Articles

Abstract IA001: Exploiting pathway activation as a new form of synthetic lethality

Abstract Targeting activated oncogenes is an effective treatment strategy across many cancers now including therapeutics targeting KRAS. Genetic events including DNA damage deficiencies and tumor suppressor mutations require alternative strategies and the concept of synthetic lethality has been applied to these alterations. PARPi inhibitors were a founding member of this class of therapeutics demonstrating enhanced activity in the setting of BRCA-deficiency. By applying large-scale loss-of-function approaches1 we and other discovered the vulnerability to PRMT5 and WRN inhibition imposed by co-deletions of MTAP and CDKN2A, and the MSI+ genotype respectively2,3. Such inhibitors are now in clinical trials. To explore the potential for paralogous genes to act as synthetic lethal gene pairs we enacted dual knockout screens. These early efforts led to the discovery of DUSP4/6 paralog dependence in the setting of BRAF and NRAS mutations in melanoma4. Intriguingly the loss of DUSP4/6 impaired cancer cell viability through the increased activation of ERK highlighting the susceptibility of cancers to pathway activation in addition to the more common sensitivity to pathway inhibition. This inappropriate activation of the ERK signaling pathway, the conflict between EGFR and KRAS activation, the synthetic lethality enacted by TRIM8 knockouts, and the effects of AR agonists on prostate cancer viability, points to a wider than expected vulnerability of cancer to inappropriate gene activation. To systematically identify context-specific gene activation induced lethalities in cancer, we developed methods for enacting gain-of-function perturbations across ∼500 barcoded cancer cell lines. With this approach, we queried the pan-cancer vulnerability landscape upon activating 10 key cancer pathway revealing activation dependencies in MAPK and PI3K pathways. Notably, we discovered novel pathway hyperactivation dependencies in subsets of APC-mutant colorectal cancers where further activation of the WNT pathway by APC knockdown or direct β-catenin overexpression led to robust antitumor effects in xenograft and patient-derived organoid models. These latter discoveries paradoxically point to the residual activity of the APC ubiquitin-ligase complex as a target in APC-mutant CRC5. 1. McDonald, E. R., 3rd et al. Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening. Cell 170, 577-592.e10 (2017). 2. Mavrakis, K. J. et al. Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to dependence on PRMT5. Science 351, 1208–1213 (2016). 3. Chan, E. M. et al. WRN helicase is a synthetic lethal target in microsatellite unstable cancers. Nature 568, 551–556 (2019). 4. Ito, T. et al. Paralog knockout profiling identifies DUSP4 and DUSP6 as a digenic dependence in MAPK pathway-driven cancers. Nat. Genet. 53, 1664–1672 (2021). 5. Chang, L. et al. Systematic profiling of conditional pathway activation identifies contextdependent synthetic lethalities. Nat. Genet. 55, 1709–1720 (2023). Citation Format: William R. Sellers. Exploiting pathway activation as a new form of synthetic lethality [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA001.

Abstract PR012: KAT6A/B and Menin-MLL complexes coordinately regulate estrogen receptor-driven gene expression programs in breast cancer

Abstract The estrogen receptor (ER) is expressed in ∼70% of breast cancers where it functions as a critical transcription factor that regulates cell growth and tumor progression. ER inhibition is a mainstay treatment for ER-positive (ER+) breast cancer, but despite the clinical benefit of targeting ER signaling, breast cancer remains the second leading cause of cancer-related death in women, with ∼50% of deaths resulting from ER+ tumors. While recurrent and metastatic ER+ tumors are typically resistant to endocrine therapies they continue to rely on ER signaling suggesting that disruption of other chromatin associated complexes that mediate ER-driven gene expression may present an alternative therapeutic strategy. One chromatin modifier important in regulating ER function is the histone acetyltransferase KAT6A. KAT6A is amplified and/or overexpressed in ∼15% of ER+ tumors and a novel KAT6A/B inhibitor has entered clinical trials for advanced breast cancer. However, as with most single agent therapies, compensatory mechanisms may attenuate the effects of KAT6A/B inhibition on ER-driven gene expression. We hypothesized that the dual targeting of distinct chromatin complexes would enhance the effects of KAT6A/B inhibition, by altering chromatin state and transcriptional output. To that end we performed an epigenetic focused CRISPR-Cas9-based functional genetic screen to identify chromatin-associated proteins that enhance KAT6A/B inhibition. CRISPR guides targeting MEN1 (encoding Menin) were among our top hits as sensitizers to treatment with the KAT6A/B inhibitor PF-9363. We confirmed that co-treatment of PF-9363, and the Menin inhibitor, SNDX-5613, had strong synergistic anti-proliferative effects in a panel of ER+ breast cancer cell lines. Mechanistically, our data revealed that KAT6A/B and Menin cooperatively regulate ER-driven gene expression and chromatin accessibility, both through direct effects on ESR1 expression as well as through effects at ER target genes. Specifically, we found that KAT6A and Menin-KMT2A complexes co-localized at promoters of ER target genes and treatment with KAT6A/B or Menin inhibitors displaced KAT6A and Menin/KMT2A from ER target genes, respectively. Yet only the combination effectively displaced both complexes from chromatin, which produced more dramatic changes in ER-directed gene expression than either small molecule alone. Combined displacement of KAT6A and Menin/KMT2A at ER-regulated genes induced loss of an active chromatin landscape characterized by reduction of activating histone modifications and loss of RNA Polymerase II binding to ER target loci. Importantly, the combination of KAT6A/B and Menin inhibition was effective in a panel of ER+ patient-derived organoid models as well as in various models of endocrine resistance, including models with ESR1 mutations, NF1 loss, and FOXA1 mutations. Both KAT6A/B and Menin inhibitors are in Phase I/II clinical trials and have shown manageable toxicity profiles to date. This combination represents a novel potential therapeutic combination for ER+ breast cancer. Citation Format: Sarah Naomi Olsen, Bryn Anderson, Charles Hatton, Rinath Jeselsohn, Myles Brown, Eneda Toska, Scott Armstrong. KAT6A/B and Menin-MLL complexes coordinately regulate estrogen receptor-driven gene expression programs in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR012.

Patient-derived parotid gland pleomorphic adenoma organoid model and its preliminary histological and functional characterization

Objective: To establish patient-derived organoid models of pleomorphic adenomas (PA) of the parotid gland and preliminarily characterize their histology, related biomarkers and functions. Methods: Fresh tumor tissue specimens were collected from surgical procedures of Oral and Maxillofacial Department. The harvested tissues were processed and cultured in a head and neck tumor organoid culture system to establish organoid models from parotid gland pleomorphic adenomas. The in vitro growth of PA organoids was recorded by light microscopy. The successfully established organoids were passaged and cryopreserved, and the cryopreserved PA organoids were revived and re-cultured to observe their viability and organoid regeneration ability. Histological characterization, as well as characterization and detection of related markers and functional proteins, were performed on the organoids, comparing them with the patient-derived tissues. Results: The constructed organoid model of pleomorphic adenoma exhibited a dense and compact three-dimensional spherical structure. Hematoxylin and eosin staining indicated morphological similarities between the organoid and its tissue of origin. Immunohistochemistry showed positive cytoplasmic staining for Calponin, cytokeratin 7, and epithelial membrane antigen in both the organoid and the source tumor tissue, suggesting consistent histopathological characteristics between the organoid and its tissue of origin. Periodic acid-Schiff staining of the organoid showed positive staining for glycogen, with positive staining located in the interior and periphery of the organoid, indicating that the organoid possessed secretory functions like the salivary gland. Conclusions: This study successfully constructed organoids of pleomorphic adenoma derived from patient samples. This model faithfully replicates the tissue morphology and biomarkers of the source tissue and exhibits biological functions associated with mucus secretion. It serves as a valuable in vitro model for studying the development and progression of salivary gland tumors.

Genome-wide CRISPR screening identifies the pivotal role of ANKRD42 in colorectal cancer metastasis through EMT regulation.

Colorectal cancer (CRC), a pervasive and lethal malignancy of gastrointestinal cancer, imposes significant challenges due to the occurrence of distant metastasis in advanced stages. Understanding the intricate regulatory mechanisms driving CRC distant metastasis is of paramount importance. CRISPR-Cas9 screening has emerged as a powerful tool for investigating tumor initiation and progression. However, its application in studying CRC distant metastasis remains largely unexplored. To establish a model that faithfully recapitulates CRC liver metastasis in patients, we developed an in vivo genome-wide CRISPR-Cas9 screening approach using a spleen-injected liver metastasis mouse model. Through comprehensive screening of a whole-genome sgRNA library, we identified ANKRD42 as a pivotal regulatory gene facilitating CRC liver metastasis. Analysis of the TCGA database and our clinical cohorts unveiled heightened ANKRD42 expression in metastases. At the cellular level, the attenuation of ANKRD42 impaired the migration and invasion processes of tumor cells. In vivo experiments further validated these observations, highlighting the diminished liver metastatic capacity of tumor cells upon ANKRD42 knockdown. To unravel the specific mechanisms by which ANKRD42 regulates CRC distant metastasis, we leveraged patient-derived organoid (PDO) models. Depleting ANKRD42 in PDOs sourced from liver metastases precipitated the downregulation of pivotal genes linked to epithelial-mesenchymal transition (EMT), including CDH2 and SNAI2, thereby effectively suppressing tumor metastasis. This study not only establishes a conceptual framework but also identifies potential therapeutic avenues for advanced-stage distant metastasis in CRC patients.

Translational Utility of Organoid Models for Biomedical Research on Gastrointestinal Diseases.

Organoid models have recently been utilized to study 3D human-derived tissue systems to uncover tissuearchitecture and adultstem cellbiology. Patient-derived organoids unambiguously provide the most suitable in vitro system to study disease biology with the actual genetic background. With the advent of much improved and innovative approaches, patient-derived organoids can potentially be used in regenerative medicine. Various human tissues were explored to develop organoids due to their multifold advantage over the conventional in vitro cell line culture approach and in vivo models. Gastrointestinal (GI) tissues have been widely studied to establish organoids and organ-on-chip for screening drugs, nutraceuticals, and other small molecules having therapeutic potential. The function of channel proteins, transporters, and transmembrane proteins was also explained. The successful application of genome editing in organoids using the CRISPR-Cas approach has been reported recently. GI diseases such as Celiac disease (CeD), Inflammatory bowel disease (IBD), and common GI cancers have been investigated using several patient-derived organoid models. Recent advancements on organoid bio-banking and 3D bio-printing contributed significantly in personalized disease management and therapeutics. This article reviews the available literature on investigations and translational applications of patient-derived GI organoid models, notably on elucidating gut-microbial interaction and epigenetic modifications.

Abstract A009: Integrative drug screening and multi-omic characterization of patient-derived bladder cancer organoids reveals novel molecular correlates of chemotherapy response

Abstract Introduction: Predicting response to therapy for each patient’s tumor is critical to improving long-term outcomes for muscle-invasive bladder cancer (MIBC). Our objective is to establish ex vivo bladder cancer patient-derived organoid (PDO) models that are representative of patients’ tumors and determine potential efficacy of standard-of-care and curated experimental therapies. Methods: Tumor material was prospectively collected from consented patients with bladder cancer to generate short-term PDO models, which were screened against a panel of clinically relevant drugs in ex vivo 3D culture. Drugs were tested at the maximum plasma concentration (Cmax) in human trials, so as to provide physiologic relevance. When material was sufficient, cells were additionally tested using full dose response. The number of drugs screened was dependent upon amount of tissue available, with up to 34 drugs screened at Cmax and 9 drugs screened in dose response format. Multiomic profiling was utilized to validate the PDO models, establish the molecular characteristics of each tumor, assess gene expression (GEX) patterns between paired primary tissue and PDOs, and identify potential biomarkers of drug response using Pearson correlation coefficients and Kruskal-Wallis tests with Dunn’s post-hoc pairwise comparison testing. Results: 106 tumors were collected from 97 patients, with 65 samples yielding sufficient material for complete multiomic molecular characterization and PDO screening with 6 to 32 drugs/combinations. RNA sequencing and GSEA enriched pathways across organoids were compared to other published datasets, noting high levels of correlation between our organoid models and in particular the Cancer Cell Line Encyclopedia urethral lines. Moreover, analysis of subtype and mutation/copy number data further indicate that our organoid dataset is representative of the full spectrum of disease. RNA sequencing and subtyping of cultured organoids compared to patient tissue indicate that organoid models are representative of patient tissue and do not undergo subtype shifts in our short-term 3D cultures. Our drug screening results highlight the large spectrum of response to chemotherapies that in fact, clinically benefit only a small proportion of patients in the neoadjuvant setting. Utilizing an integrative approach, novel correlations between ex vivo drug responses and genomic alterations, GEX, and protein expression were identified, including a multiomic signature of gemcitabine response. Conclusions: Rapid organoid development, characterization, and drug screening allows for the prediction of therapeutic response in ~10 days following sample collection. Use of this technique on tissue provided during disease work-up may further guide selection of effective therapeutic agents in patients with bladder cancer, beyond the typical unranked lists derived from genomic analyses. This work would help maximize the overall benefits of standard of care therapies and could be used to identify alternative therapeutics for patients who progress on standard therapies. Citation Format: Nathan M. Merrill, Samuel D. Kaffenberger, Liwei Bao, Nathalie Vandecan, Laura E. Goo, Athena Apfel, Xu Cheng, Zhaoping Qin, Chia-Jen Liu, Armand Bankhead, Yin Wang, Varun Kathawate, Lila Tudrick, Habib Serhan, Zackariah Farah, Chad Ellimoottil, Khaled S. Hafez, Lindsey A. Herrel, Jeffrey S. Montgomery, Todd M. Morgan, Simpa S. Salami, Alon Z. Weizer, Peter J. Ulintz, Mark L. Day, Matthew B. Soellner, Phillip L. Palmbos, Sofia D. Merajver, Aaron M. Udager. Integrative drug screening and multi-omic characterization of patient-derived bladder cancer organoids reveals novel molecular correlates of chemotherapy response [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A009.

Abstract A028: A living biobank of patient-derived organoids reveals a role for KMT2D in maintaining luminal identity in urothelial carcinoma

Abstract Patient-derived organoid models provide a highly amenable experimental platform to study human tumor biology. Over the past seven years, our group has generated a living biobank of 82 organoid lines established from urothelial carcinomas. These organoids have been generated from all stages, are capable of unlimited expansion in vitro, and recapitulate histopathological and molecular features of their corresponding primary tumors. In addition, we have characterized the genomic, transcriptomic, and chromatin accessibility profiles of these organoid lines. Using these lines, we have identified a novel form of lineage plasticity in which a subset of organoids derived from luminal NMIBC tumors can acquire basal features in culture, but reverse back to a luminal phenotype following orthotopic xenografting in immunodeficient mice. We have focused our studies on this luminal-to-basal (L-B) plasticity since the acquisition of basal phenotypes is linked to secondary MIBC. In ongoing studies, we have examined the genomic correlates of L-B plasticity and found that loss-of-function (LOF) mutations in the epigenetic regulator KMT2D are enriched in plastic lines derived from NMIBC and in stable basal lines from basal/squamous MIBC. We also found that tumors with squamous histology in the TCGA cohort are enriched for KMT2D LOF mutations. KMT2D encodes a histone methyltransferase that adds H3K4me1 active histone marks to enhancer regions and is frequently mutated in urothelial cancers. To investigate its functional role, we performed CRISPR knock-out of KMT2D in a stable luminal organoid line and found that isogenic knock-out organoids acquired basal phenotypes. Using a barcoded lineage-tracing approach known as CellTag, we have found that acquisition of basal phenotypes occurs in a clonal fashion at the single-cell level. In parallel, we have performed a small molecule screen to identify compounds that can reverse L-B plasticity, using 17 compounds that target a range of epigenetic regulators. We found that GSK-LSD1, an inhibitor of KDM1A, can restore luminal phenotypes to plastic organoid lines in culture and increase the genomic distribution of the H3K4me1 mark, as detected by CUT&Tag. Interestingly, KDM1A is a histone demethylase that removes H3K4me1/2 marks, and thus can antagonize KMT2D. Using CellTag, we found that reversal of the basal phenotype occurs in a non-clonal fashion and represents a cell state transition at the level of single cells. In further studies, we have shown that KDM1A inhibitor treatment of fresh patient samples can prevent the L-B transition. We have also demonstrated in an organoid outgrowth assay that KDM1A inhibition can prevent invasive behavior in vitro. Moreover, an orally available KDM1A inhibitor, ORY-1001, can also reverse phenotypic plasticity, and we are currently testing its activity in xenografts to determine whether it can reverse invasive phenotypes. Overall, these results demonstrate the utility of patient-derived organoid models to investigate novel features of bladder cancer and develop therapeutic approaches. Citation Format: John R. Christin, Alana Nguyen, Talal Syed, Kwanghee Kim, Clementine Le Coz, Caroline J. Laplaca, Luis A. Pina, Reuben Akabas, Hanina Hibshoosh, Andrew T. Lenis, Guarionex J. Decastro, Christopher B. Anderson, James M. McKiernan, Chao Lu, Hikmat A. Al-Ahmadie, David B. Solit, Michael M. Shen. A living biobank of patient-derived organoids reveals a role for KMT2D in maintaining luminal identity in urothelial carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A028.

Preclinical efficacy of CBR-5884 against epithelial ovarian cancer cells by targeting the serine synthesis pathway

Reprogramming of the serine synthesis pathway (SSP) is intricately linked to the progression of epithelial ovarian cancer (EOC). CBR-5884, a selective small-molecule inhibitor targeting phosphoglycerate dehydrogenase (PHGDH), effectively impedes the de novo synthesis of serine within cancer cells. This study aimed to evaluate the inhibitory effect of CBR-5884 on EOC cells and delineate its specific mechanism, thereby proposing a novel therapeutic approach for treating EOC. The suppression of serine biosynthesis after CBR-5884 treatment was evaluated using RNA sequencing and a serine assay kit, and the results showed that CBR-5884 effectively downregulated serine biosynthesis in EOC cells, particularly those expressing high levels of PHGDH. In vitro studies revealed that CBR-5884 demonstrated significant antitumor effects and suppressed migration and invasion of EOC cells through down-regulation of the integrin subunit beta 4 (ITGB4)/extracellular signal-regulated kinase (ERK)/epithelial–mesenchymal transition signal axis. Additionally, CBR-5884 mitigated the stemness of EOC cells and heightened their sensitivity to chemotherapy. Moreover, in vivo studies revealed that CBR-5884 significantly delayed tumor growth, with histological analysis indicating the safety profile of CBR-5884. Finally, the patient-derived organoid (PDO) models were utilized to explore the preclinical efficacy of CBR-5884 against EOC cells, and the results unveiled that CBR-5884 impeded proliferation and downregulated the expression of ITGB4 in EOC PDO models. Our findings supports the anticancer properties of CBR-5884 in EOC cells exhibiting high PHGDH expression, manifesting through the suppression of proliferation, migration, and invasion, while enhancing chemotherapy sensitivity, suggesting that CBR-5884 holds promise as an efficacious strategy for the treatment of EOC.

Biological impact and therapeutic potential of a novel camptothecin derivative (FLQY2) in pancreatic cancer through inactivation of the PDK1/AKT/mTOR pathway

BackgroundCamptothecin (CPT), a pentacyclic alkaloid with antitumor properties, is derived from the Camptotheca acuminata. Topotecan and irinotecan (CPT derivatives) were first approved by the Food and Drug Administration for cancer treatment over 25 years ago and remain key anticancer drugs today. However, their use is often limited by clinical toxicity. Despite extensive development efforts, many of these derivatives have not succeeded clinically, particularly in their effectiveness against pancreatic cancer which remains modest. Aim of the studyThis study aimed to evaluate the therapeutic activity of FLQY2, a CPT derivative synthesized in our laboratory, against pancreatic cancer, comparing its efficacy and mechanism of action with those of established clinical drugs. MethodsThe cytotoxic effects of FLQY2 on cancer cells were assessed using an MTT assay. Patient-derived organoid (PDO) models were employed to compare the sensitivity of FLQY2 to existing clinical drugs across various cancers. The impact of FLQY2 on apoptosis and cell cycle arrest in Mia Paca-2 pancreatic cancer cells was examined through flow cytometry. Transcriptomic and proteomic analyses were conducted to explore the underlying mechanisms of FLQY2′s antitumor activity. Western blotting was used to determine the levels of proteins regulated by FLQY2. Additionally, the antitumor efficacy of FLQY2 in vivo was evaluated in a pancreatic cancer xenograft model. ResultsFLQY2 demonstrated (1) potent cytotoxicity; (2) superior tumor-suppressive activity in PDO models compared to current clinical drugs such as gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, ivosidenib, infinitinib, and lenvatinib; (3) significantly greater tumor inhibition than paclitaxel liposomes in a pancreatic cancer xenograft model; (4) robust antitumor effects, closely associated with the inhibition of the TOP I and PDK1/AKT/mTOR signaling pathways. In vitro studies revealed that FLQY2 inhibited cell proliferation, colony formation, induced apoptosis, and caused cell cycle arrest at nanomolar concentrations. Furthermore, the combination of FLQY2 and gemcitabine exhibited significant inhibitory and synergistic effects. ConclusionThe study confirmed the involvement of topoisomerase I and the PDK1/AKT/mTOR pathways in mediating the antitumor activity of FLQY2 in treating Mia Paca-2 pancreatic cancer. Therefore, FLQY2 has potential as a novel therapeutic option for patients with pancreatic cancer.

MRI radiomics captures early treatment response in patient-derived organoid endometrial cancer mouse models.

Radiomics can capture microscale information in medical images beyond what is visible to the naked human eye. Using a clinically relevant mouse model for endometrial cancer, the objective of this study was to develop and validate a radiomic signature (RS) predicting response to standard chemotherapy. Mice orthotopically implanted with a patient-derived grade 3 endometrioid endometrial cancer organoid model (O-PDX) were allocated to chemotherapy (combined paclitaxel/carboplatin, n=11) or saline/control (n=13). During tumor progression, the mice underwent weekly T2-weighted (T2w) magnetic resonance imaging (MRI). Segmentation of primary tumor volume (vMRI) allowed extraction of radiomic features from whole-volume tumor masks. A radiomic model for predicting treatment response was derived employing least absolute shrinkage and selection operator (LASSO) statistics at endpoint images in the orthotopic O-PDX (RS_O), and subsequently applied on the earlier study timepoints (RS_O at baseline, and week 1-3). For external validation, the radiomic model was tested in a separate T2w-MRI dataset on segmented whole-volume subcutaneous tumors (RS_S) from the same O-PDX model, imaged at three timepoints (baseline, day 3 and day 10/endpoint) after start of chemotherapy (n=8 tumors) or saline/control (n=8 tumors). The RS_O yielded rapidly increasing area under the receiver operating characteristic (ROC) curves (AUCs) for predicting treatment response from baseline until endpoint; AUC=0.38 (baseline); 0.80 (week 1), 0.85 (week 2), 0.96 (week 3) and 1.0 (endpoint). In comparison, vMRI yielded AUCs of 0.37 (baseline); 0.69 (w1); 0.83 (week 2); 0.92 (week 3) and 0.97 (endpoint). When tested in the external validation dataset, RS_S yielded high accuracy for predicting treatment response at day10/endpoint (AUC=0.85) and tended to yield higher AUC than vMRI (AUC=0.78, p=0.18). Neither RS_S nor vMRI predicted response at day 3 in the external validation set (AUC=0.56 for both). We have developed and validated a radiomic signature that was able to capture chemotherapeutic treatment response both in an O-PDX and in a subcutaneous endometrial cancer mouse model. This study supports the promising role of preclinical imaging including radiomic tumor profiling to assess early treatment response in endometrial cancer models.

Enhancing the Efficacy of HIPEC Through Bromelain: A Preclinical Investigation in Appendiceal Cancer

IntroductionAppendiceal cancer (AC) excessive mucin production is a barrier to heated intraperitoneal chemotherapy (HIPEC) drug delivery. Bromelain is a pineapple stem extract with mucolytic properties. We explored bromelain treatment effects against mucinous AC in a patient-derived tumor organoid (PTO) model and an AC cell line.Patients and MethodsPTOs were fabricated from tumor specimens obtained from patients with AC undergoing cytoreductive surgery with HIPEC. PTOs underwent HIPEC treatment with bromelain, cisplatin, and mitomycin C (MMC) at 37 °C and 42 °C with and without bromelain pretreatment.ResultsFrom October 2020 to May 2023, 16 specimens were collected from 13 patients with low-grade (12/16, 75%) and high-grade AC (4/16, 25%). The mucin-depleting effects of bromelain were most significant in combination with N-acetylcysteine (NAC) compared with bromelain (47% versus 10%, p = 0.0009) or NAC alone (47% versus 12.8%, p = 0.0027). Bromelain demonstrated > 31% organoid viability reduction at 60 min (p < 0.001) and > 66% in 48 h (p < 0.0001). Pretreatment with bromelain increased cytotoxicity of both cisplatin and MMC HIPEC conditions by 31.6% (p = 0.0001) and 35.5% (p = 0.0001), respectively. Ki67, CK20, and MUC2 expression decreased after bromelain treatment; while increased caspase 3/7 activity and decreased Bcl-2 (p = 0.009) and Bcl-xL (p = 0.01) suggest induction of apoptosis pathways. Furthermore, autophagy proteins LC3A/B I (p < 0.03) and II (p < 0.031) were increased; while ATG7 (p < 0.01), ATG 12 (p < 0.04), and Becline 1(p < 0.03), expression decreased in bromelain-treated PTOs.ConclusionsBromelain demonstrates cytotoxicity and mucolytic activity against appendiceal cancer organoids. As a pretreatment agent, it potentiates the cytotoxicity of multiple HIPEC regimens, potentially mediated through programmed cell death and autophagy.

Abstract PO3-28-12: Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance

Abstract Standard of care for breast cancer (BC) treatment including Selective-Estrogen Receptor (ER)-modulators and/or degraders (SERM; tamoxifen, SERD;Fulvestrant) or cyclin-dependent kinases CDK4 and CDK6 (palbociclib) can be effective therapeutic strategies. However, drug toxicity and/or drug resistance severely limit these approaches and patients succumb to the disease. Therefore, there is an urge to develop new therapeutic strategies against ER+-BC. Ion channels are general pharmacological targets however, little is known about the role of this class of proteins in cancer biology. We discovered that expression of the KCa3.1 potassium ion channel is downregulated in ER+-BC. Also, patients expressing high level of KCa3.1 present a better overall survival (OS) and relapse free survival when compared with patients with low expression. Thus, these data indicate that KCa3.1 is a prognostic factor in ER+-BC and that high activity of this channel plays a fundamental role in an oncosuppressor signature. Our central hypothesis is that pharmacological targeting KCa3.1 with activator molecules arrests growth of ER+-BC. We discovered that ER+-BC patient-derived organoids (PDO) and cell lines treated with KCa3.1 agonists including the FDA approved Chlorzoxazone, (CZX) arrests significantly inhibit tumor growth. Activation of KCa3.1 arrested the cell cycle in the G0/G1 phase by activating a senescent-like phenotype. Furthermore, our studies on the biochemical signaling that underlines this event demonstrated that increased expression level of KCa3.1 protein associated with lower expression level of ERa in a cell cycle phase-dependent manner. Furthermore, use of KCa3.1activator produced a proteasomal-dependent ERa degradation. To better characterize the therapeutic benefit of targeting the KCa3.1, we assessed the effects of CZX on developed several tamoxifen and/or palbociclib resistant cell lines and PDO models. We found that the combination of CZX strongly potentiated the lethal effects of both tamoxifen and palbociclib in the relative resistant models. We conclude that pharmacological targeting KCa3.1 potassium channel in ER+-BC can be considered a novel, safe and efficacious therapeutic strategy. Citation Format: Davide Delisi, Metin Cetin, Ozge Saatci, Ozgur Sahin, Saverio Gentile. Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance [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 PO3-28-12.

Abstract PO4-14-12: Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids

Abstract Background: Activating mutations in ERBB2 (HER2) are enriched &amp;gt;4-fold in invasive lobular carcinoma (ILC) with a rate of up to 19% in metastatic ILC. ILC is a histologic subtype of breast cancer characterized by loss of E-cadherin (CDH1), suggesting a potential interaction between loss of CDH1 and mutations in ERBB2. Recent trials have demonstrated promising single agent efficacy using the irreversible pan-HER tyrosine kinase inhibitor (TKI), neratinib, in patients with metastatic ERBB2 mutant ILC. However, further studies on combination therapies with other anticancer agents are needed to increase response rate and progression free survival for these patients. HER2-targeted antibody drug conjugates (ADC), particularly trastuzumab deruxtecan (T-DXd), have shown great promise in HER2-low metastatic breast cancer. Yet, their efficacy as a single agent or with HER2 TKIs in HER2-low and -mutant ILC is unknown and warrants investigation. Methods: Past studies analyzing ERBB2 mutations used overexpression of ERBB2 mutant cDNA, but this approach does not faithfully recapitulate the human disease. To model ERBB2 missense mutations as found in human breast cancers, we used CRISPR-based prime editing to generate a panel of isogenic ILC cell lines and patient-derived organoids (PDO) harboring ERBB2 wild-type (WT) or ERBB2 mutations (S310F or V777L). Both of these ERBB2 mutations have been previously characterized and are known to be activating mutations. We then used them to test neratinib and other TKIs with ADCs, including T-DXd and trastuzumab emtansine (T-DM1). Results: We successfully introduced single copy, heterozygous activating ERBB2 mutations (S310F or V777L) into two ERBB2-nonamplified metastatic ILC cell lines (MDA-MB-134 and SUM44PE) and one ERBB2-nonamplified metastatic ILC PDO (IPM-BO-053). Positive clones carrying the mutations were verified by Sanger sequencing and droplet digital PCR and subsequently pooled together. We further demonstrated that these mutations hyperactivated HER2 and downstream signaling pathways. ILC cell lines harboring these mutations showed enhanced sensitivity to HER2 TKIs but not ADCs. In contrary, ERBB2 mutations did not alter responses of IPM-BO-053 PDOs to HER2 TKIs but significantly increased responses to ADCs. Interestingly, we also observed accelerated HER2 protein degradation upon heregulin stimulation in ERBB2 mutant ILC PDOs, suggesting activating ERBB2 mutations may enhance ADC/HER2 complex internalization, degradation, and release of payloads. Lastly, we explored drug synergy between HER2 TKIs and ADCs in isogenic IPM-BO-053 PDOs and found that combination of T-DXd and neratinib or afatinib showed synergy (Combination Index &amp;lt; 1). Conclusions: Although the reason for the discrepancies in drug response between ILC cell lines and PDOs is not clear, we hypothesize that response in 3D PDOs might be more faithfully representing response seen in patients. We will generate additional ILC PDOs with knock-in ERBB2 mutations to validate our findings. Irreversible HER2 TKIs, such as neratinib and afatinib, showed synergy with T-DXd in ERBB2 mutant ILC PDOs. This holds important therapeutic implications in light of current treatment options for ILC. In future experiments, we will test if neratinib or afatinib increases endocytic uptake of T-DXd using a fluorescently labeled endocytosis tracker. Further, an in-depth molecular characterization of our isogenic cell lines and PDOs models is ongoing to gain mechanistic insights into how activating ERBB2 mutations increase HER2 internalization and degradation. Our in vitro studies provide a strong foundation for in vivo testing of neratinib or afatinib with T-DXd for ERBB2 mutant ILC. Citation Format: Jie Bin Liu, Danielle Tseng, Jagmohan Hooda, Daniel Brown, Adrian Lee, Steffi Oesterreich. Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids [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 PO4-14-12.

Abstract PS09-09: Multiomics profiling and molecular classification refine precision treatment strategies for HER2-positive breast cancer

Abstract Background: Anti-HER2 targeted therapy has achieved a series of breakthroughs. However, the current treatment strategy regarding HER2-positive breast cancer remains indiscriminate and lacks specificity, which limits the further improvement of overall treatment response and may lead to overtreatment and extra cost for some patients. Our study aims to reveal the molecular heterogeneity of HER2-positive breast cancer to guide a more precise treatment. Patients and methods: We selected HER2-positive breast cancer patients treated at Fudan University Shanghai Cancer Center between 2013 and 2014 and conducted genomic, transcriptomic, proteomic and metabolomic profiling. We then applied a non-negative matrix factorization algorithm on transcriptomic data to obtain an unsupervised classification. And we further studied the correlation between subtypes and corresponding treatment strategies in multiple cohorts of adjuvant and neoadjuvant therapy. For clinical accessibility, we developed convolutional neural network models through deep learning algorithm based on digital pathology to identify different subtypes. Additionally, we explored novel treatment strategies using the patient-derived organoids (PDOs) models. Results: We established a novel multiomics cohort of 180 HER2- breast cancer patients and classified them into four clinically significant molecular subtypes: (1) A classical HER2-enriched (HER2-CLA, N=51) subtype characterized by strong ERBB2 signaling and remarkable sensitivity to anti-HER2-targeted therapy (pathologic complete response with dual-targeted therapy: 93%). (2) an immunomodulatory (HER2-IM, N=36) subtype characterized by an immune-activated microenvironment and excellent prognosis with current treatment (no relapse in 97% of patients with a median follow-up of 86 months). Tumors of this subtype were therefore candidates for de-escalatory treatment. (3) A luminal-like (HER2-LUM, N=55) subtype distinguished by activated estrogen receptor signaling and (4) a basal/mesenchymal-like (HER2-BM, N=38) subtype enriched in activated receptor tyrosine kinase pathways. HER2-LUM and HER2- BM showed limited benefit from anti-HER2 therapy, and thus, add-on therapies might be needed. The overall area under the curve (AUC) of the convolutional neural network model based on digital pathology for identifying different subtypes is 0.77. In the exploration of novel treatment strategies, we found in the PDO model that the HER2-LUM subtype is more sensitive to a treatment regimen combining standard (chemotherapy and targeted therapy) with subsequent endocrine therapy and CDK4/6 inhibitors compared to other subtypes. Additionally, the HER2-BM subtype demonstrated greater sensitivity to treatment with a combination of EGFR inhibitors, PDGFR inhibitors or VEGFR inhibitors. Conclusion: We uncovered a high degree of molecular heterogeneity in HER2-positive breast cancer and illustrated its impact on treatment response. More precise treatment can be given according to the characteristics of different subtypes, which may achieve good efficacy and simultaneously reduce overtreatment and extra cost. The comprehensive profiling of HER2-positive breast cancers could also serve as an important resource for further exploration. Key Words: HER2-positive breast cancer cohort; molecular classification; targeted therapy; precision treatment; de-escalatory treatment. Citation Format: Yu-Wei Li, Ding Ma, Xiang-Rong Wu, Lei-Jie Dai, Shen Zhao, Yu-Zheng Xu, Xi Jin, Xiao Yi, Ying Wang, Cai-Jin Lin, Yi-Fan Zhou, Tong Fu, Wen-Tao Yang, Ming Li, Hong Lv, Yi-Zhou Jiang, Zhi-Ming Shao. Multiomics profiling and molecular classification refine precision treatment strategies for HER2-positive breast cancer [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 PS09-09.

Abstract PO1-06-07: EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer

Abstract PO1-06-07: EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer

Abstract PO2-23-08: High intratumor heterogeneity induced PSME2 confers endocrine resistance via ERα PARylation in hormone receptor-positive and HER2-negative breast cancer

Abstract Background: Hormone receptor-positive and HER2-negative breast cancer, characterized by its significant intratumor heterogeneity (ITH), poses increased recurrence, metastasis risk, and endocrine resistance. A comprehensive understanding of the genomic and biological characteristics underlying ITH is crucial for the development of effective therapeutic strategies. Methods: We leveraged the multiomics data from our large cohort of 381 hormone receptor-positive and HER2-negative breast tumors, supplemented with single-cell sequencing (scRNA-seq) data from 9 individual samples, to investigate ITH and its associated molecular features. Mechanistic investigations were conducted using Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) proteomics, co-immunoprecipitation (Co-IP), Chromatin Immunoprecipitation followed by quantitative PCR (ChIP-qPCR), and dual-luciferase reporter assays. We specifically focused on the role of Proteasome activator subunit beta (PSME2), a potential regulator of drug resistance and metastasis. Results: We quantified the degree of ITH at sample level using multi-omics and scRNA-seq data and observed a correlation between higher ITH levels, increased genomic instability, upregulation of the STING type-I-interferon-dependent pathway, and poor efficacy of endocrine therapy. Both bulk and single-cell data revealed an enrichment of PSME2, associated with type-I interferon, in groups exhibiting high ITH. Patients with elevated PSME2 expression manifested a poorer prognosis and diminished responsiveness to endocrine therapy. Within the context of ITH, we discovered that IFN-I transcriptionally activated PSME2 via STAT1. Further investigation revealed a complex interplay among PSME2, PARP1, and EWS, wherein PSME2-mediated degradation of EWS relieved the transcriptional repression of PARP1, resulting in its overexpression. This series of events, orchestrated by PSME2, led to ERα PARylation and promoted tamoxifen resistance. In subsequent clinical translation studies employing mouse xenograft, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models, we demonstrated that PARP1 inhibitors could effectively mitigate tamoxifen resistance induced by high PSME2 expression, providing new insights into therapeutic strategies. Conclusions: Our study highlights the importance of understanding ITH in hormone receptor-positive and HER2-negative breast cancer and identifies PSME2 as a key player in endocrine resistance and patient prognosis. The application of PARP1 inhibitors shows promise in improving treatment outcomes in tumors with elevated ITH and PSME2 expression. Working Modle Citation Format: Li-Ping Ge, Zi-Yu Wang, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. High intratumor heterogeneity induced PSME2 confers endocrine resistance via ERα PARylation in hormone receptor-positive and HER2-negative breast cancer [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 PO2-23-08.

Exploring Contribution of HMGA2 to the Aggressive Tumor Structure in Urothelial Carcinoma to Identify Prospective Clinical Applications

For patients with advanced upper tract urothelial carcinoma (UTUC), the five-year survival rate is approximately 20-40%. In previous real-world data analyses, we observed that UTUC patients with a sessile tumor architecture tend to exhibit more aggressive behavior and concurrently have poorer outcomes in terms of metastasis-free and cancer-specific survival. This suggests that tumor architecture may serve as a crucial prognostic factor for predicting outcomes. We utilized various tumor tissue specimens, categorizing them based on tumor architecture into papillary and sessile groups. RNA sequencing was used to analyze the two groups, and significant differences in the expression levels of high mobility group AT-hook 2 (HMGA2) were found. In the more aggressive sessile tumor architecture, HMGA2 exhibited higher expression levels. HMGA2 is currently known to participate in various biological processes, including the cell cycle, DNA damage repair, apoptosis, and epithelial-mesenchymal transition. Additionally, HMGA2 is abnormally regulated in several human cancers, such as lung, breast, ovarian, and bladder cancers. Elevated expression of HMGA2 is associated with a higher risk of tumor recurrence and cancer progression. Hence, we hypothesize that HMGA2 may regulate tumor cell epithelial-mesenchymal transition through the PI3K-Akt cell signaling pathway, influencing tumor architecture and cancer prognosis. Attempting to downregulate HMGA2 should theoretically enhance the effectiveness of chemotherapeutic drugs in achieving an anti-cancer effect. We discovered that inhibiting the expression of HMGA2 using siRNA in UTUC cells, such as BFTC909, significantly reduces cell proliferation and migration. Moreover, the addition of an HMGA2 inhibitor known as suramin enhances cytotoxic effects and decreases cell migration. Our preliminary findings provide evidence indicating a correlation between high levels of HMGA2 expression and aggressive tumor architecture in UTUC. We designed UTUC patient-derived organoids (PDO). The test results revealed that the organoids, as indicated by HE stain and Ki67 immunohistochemical (IHC) staining, closely resembled their corresponding parental tumors. Currently, it appears that the organoids have retained the original tumor structure and protein molecular expression characteristics. The PDO model is hoped to serve as a valuable tool in aiding the selection of suitable therapeutic strategies as clinical applications. This study was funded by the Chang Gung Medical Foundation, Taiwan (CMRPG8M0131, and CMRPG8M0132) and the Ministry of Science and Technology, Taiwan (MOST 111-2314-B-182A-140). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The combination of breast cancer PDO and mini-PDX platform for drug screening and individualized treatment.

The majority of advanced breast cancers exhibit strong aggressiveness, heterogeneity, and drug resistance, and currently, the lack of effective treatment strategies is one of the main challenges that cancer research must face. Therefore, developing a feasible preclinical model to explore tailored treatments for refractory breast cancer is urgently needed. We established organoid biobanks from 17 patients with breast cancer and characterized them by immunohistochemistry (IHC) and next generation sequencing (NGS). In addition, we in the first combination of patient-derived organoids (PDOs) with mini-patient-derived xenografts (Mini-PDXs) for the rapid and precise screening of drug sensitivity. We confirmed that breast cancer organoids are a high-fidelity three-dimension (3D) model invitro that recapitulates the original tumour's histological and genetic features. In addition, for a heavily pretreated patient with advanced drug-resistant breast cancer, we combined PDO and Mini-PDX models to identify potentially effective combinations of therapeutic agents for this patient who were alpelisib + fulvestrant. In the drug sensitivity experiment of organoids, we observed changes in the PI3K/AKT/mTOR signalling axis and oestrogen receptor (ER) protein expression levels, which further verified the reliability of the screening results. Our study demonstrates that the PDO combined with mini-PDX model offers a rapid and precise drug screening platform that holds promise for personalized medicine, improving patient outcomes and addressing the urgent need for effective therapies in advanced breast cancer.

Inhibition of the transmembrane transporter ABCB1 overcomes resistance to doxorubicin in patient-derived organoid models of HCC.

Transarterial chemoembolization is the first-line treatment for intermediate-stage HCC. However, the response rate to transarterial chemoembolization varies, and the molecular mechanisms underlying variable responses are poorly understood. Patient-derived hepatocellular carcinoma organoids (HCCOs) offer a novel platform to investigate the molecular mechanisms underlying doxorubicin resistance. We evaluated the effects of hypoxia and doxorubicin on cell viability and cell cycle distribution in 20 patient-derived HCCO lines. The determinants of doxorubicin response were identified by comparing the transcriptomes of sensitive to resistant HCCOs. Candidate genes were validated by pharmacological inhibition. Hypoxia reduced the proliferation of HCCOs and increased the number of cells in the G0/G1 phase of the cell cycle, while decreasing the number in the S phase. The IC50s of the doxorubicin response varied widely, from 29nM to >1µM. Doxorubicin and hypoxia did not exhibit synergistic effects but were additive in some HCCOs. Doxorubicin reduced the number of cells in the G0/G1 and S phases and increased the number in the G2 phase under both normoxia and hypoxia. Genes related to drug metabolism and export, most notably ABCB1, were differentially expressed between doxorubicin-resistant and doxorubicin-sensitive HCCOs. Small molecule inhibition of ABCB1 increased intracellular doxorubicin levels and decreased drug tolerance in resistant HCCOs. The inhibitory effects of doxorubicin treatment and hypoxia on HCCO proliferation are variable, suggesting an important role of tumor-cell intrinsic properties in doxorubicin resistance. ABCB1 is a determinant of doxorubicin response in HCCOs. Combination treatment of doxorubicin and ABCB1 inhibition may increase the response rate to transarterial chemoembolization.

Comparison of Surgical and Colonoscopy Tissue to Establish Colorectal Patient-derived Organoids.

Patient-derived organoids (PDOs) are ex vivo models that retain the functions and characteristics of individualized source tissues, including a simulated tumor microenvironment. However, the potential impact of undiscovered differences between tissue sources on PDO growth and progression remains unclear. This study aimed to compare the growth and condition of PDO models originating from surgical resection and colonoscopy and to provide practical insights for PDO studies. Tissue samples and relevant patient clinical information were collected to establish organoid models. PDOs were derived from both surgical and colonoscopy tissues. The growth of the organoids, including their state, size, and success rate of establishment, was recorded and analyzed. The activity of the organoids at the end stage of growth was detected using calcein-AM fluorescence staining. The results showed that the early growth phase of 2/3 colonoscopy-derived organoids was faster compared to surgical PDOs, with a growth difference observed within 11-13 days of establishment. However, colonoscopy-derived organoids exhibited a diminished growth trend after this time. There were no significant differences observed in the terminal area and quantity between the two types of tissue-derived organoids. Immunofluorescence assays of the PDOs revealed that the surgical PDOs possessed a denser cell mass with relatively higher viability than colonoscopy-derived PDOs. In the establishment of colorectal patient-derived organoids, surgically derived organoids require a slightly longer establishment period, while colonoscopy-derived organoids should be passaged prior to growth inhibition to preserve organoid viability.