Ethan Samuel Lin

Patient-derived organoids (PDOs) have transformed translational cancer research, allowing tractable models that better represent clinical features than traditional immortalized cell lines. Here we describe two PDOs with differential responses to carboplatin derived from sequential ascites fluid collections from a patient with high-grade müllerian carcinoma, that could not be further subclassified on the omental biopsy. Uterine origin was clinically excluded by pelvic imaging/CT scan of the uterus and absence of vaginal bleeding. Successful derivation from independent collections enabled comparison of intra-patient heterogeneity across sequential ascites samples and demonstrates that PDO efficiency rate is at least partly patient-specific or tumor-dependent. We performed long-read whole genome sequencing on the two PDOs, OC104 and OC109, to better characterize the structural variant landscape while also obtaining information on single nucleotide variants and DNA methylation. In addition to confirming single nucleotide variants noted in clinical sequencing (TP53, KRAS, SPOP, PPP2R1A, KMT2D), we identified additional variants in TSC2, NCOR2, and CTNNA2 that are predicted to be likely pathogenic. The spectrum of mutations, particularly the coincident KRAS and TP53, highlighted unexpected overlap with ovarian mucinous carcinoma. We also identified larger insertions and deletions that result in non-synonymous variants in MUC5AC, TPRX1, and BMX, as well as four translocation events, including two that could not have been resolved with short-read sequencing. Differentially methylated promoters between the two PDOs include 201 oncogenes and tumor suppressor genes, with HNF1A, MSI2, and SETBP1 having methylation directions consistent with these genes’ roles in platinum response differences observed between the PDOs. Notably, the clonal nature of PDOs produced from two samples taken one week apart is important for the field to appreciate, particularly since they have clonal differences in platinum response. The temporal differences in clonality may indicate a limitation of low volume sampling, however may provide opportunity to longitudinally predict clinical outcomes. We also demonstrate the ability of long-read sequencing to add detail into the genomics and epigenetics of ovarian cancer.

Higher-order chromatin structures (HOCS) are fundamental to genome organization, gene regulation, and cellular homeostasis. This review examines the epigenetic mechanisms shaping HOCS, including DNA methylation, histone modifications, chromatin remodeling, and RNA-based regulatory processes. We also discuss the role of architectural proteins in maintaining chromatin topology while allowing dynamic changes to chromatin structure, thereby influencing gene expression. Growing evidence indicates that disruptions in HOCS contribute to a diverse array of human diseases, including cancer, aging-related disorders, and congenital abnormalities, primarily through aberrant gene regulation. We further discuss the concept of distinct genomic areas, in which specific chromatin regions orchestrate three-dimensional (3D) genome dynamics, positioning them as potential biomarkers and therapeutic targets. By emphasizing chromatin architecture on a global scale rather than at the level of individual genes, this review underscores its emerging relevance to precision medicine. Finally, we synthesize current technical advances, outline future directions for leveraging chromatin topology in disease diagnosis and treatment, and highlight key biological insights to reshape our understanding of genome function.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer mortality in the United States (US) with a 10% 5-year survival. Acquired resistance plays a critical role in progression, however targeted therapeutics have shown minimal clinical activity. There remains an unmet need to provide combination targeted therapies across the breadth of clinical development. KinderMiner is a highly efficient text mining algorithm developed to predict association with search terms across publicly available research. BMS986158 is a novel bromodomain and extraterminal (BET) inhibitor in early phase clinical trials, recently reported to have preclinical activity in combination with chemotherapy. Methods: Potential genes for targeting in combination with BMS986158 were queried using KinderMiner (KM) and Serial KinderMiner (SKiM). Search terms included synonymous terms of “drug resistance” and “BET”. BMS986158 was assayed for single agent activity at 72h and 144h. Lead agents in active clinical trials were screened for synergistic activity. All assays were performed with patient derived organoids (PCOs) in a low volume format with high content imaging, using dose titration in combination grid to assay synergy. Viability was assessed using 3D CellTiterGlo (CTG, Promega Inc) (50% v/v) with synergy scores determined by SynergyFinder 3.0 for Bliss Independence (Bliss), Highest Single Agent (HSA), and Loewe Additivity (Loewe). Results: A KM/SKiM query was performed against >30M primary literature reports resulting in 39 unique genes with significant relationship with BET. CDK9 was selected as a lead gene target based on CDK9 inhibitor, AZD4573, in active clinical trials with highly significant prediction from text mining screen (p50 activity across multiple primary PDAC PCOs (n=5) that improved from 72h [43-411nM] in comparison to 144h [12-54nM]. AZD4573 was found to have single agent IC₅₀ ranging from 18-246nM across 3 primary cultures. Therapeutically relevant dosing of 6.25nM BMS986158 and 12.5nM AZD4573 in combination was found to induce significant response including normalized change in diameter relative to control, single agent BMS986158, and AZD4573 (effect sizes=1.7, 1.4, 0.7) The combination of BMS986158 and AZD4573 was also found to have augmented activity with positive synergy scores across all reference models: Bliss=3.47, HSA=8.81, and Loewe=15.92. Conclusion: Here, we adapt a novel text-mining algorithm, KinderMiner, to screen agents for combination with BMS986158 to inform combination therapeutics. The combination of BMS986158 and AZD4573 was shown to have improved activity in a model of PDAC PCOs. Validation of the combination efficacy across different PDAC PCOs is ongoing to compare this activity across a diversity of mutational profiles. Future directions will assay the mechanistic modeling to characterize the interaction between transcriptional regulation and the induction of apoptosis. Citation Format: Austin Stram, Luke Koeppel, Rob Millikin, Eleanor Riedl, Md Shahadat Hossan, Ethan S Lin, Ron Stewart, Jeremy D Kratz. Activity of dual BET and CDK9 inhibition in Pancreatic Ductal Adenocarcinoma informed from KinderMiner Prediction [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A164.

Pancreatic and ampullary cancers remain highly morbid diseases for which accurate clinical predictions are needed for precise therapeutic predictions. Patient-derived cancer organoids have been widely adopted; however, prior work has focused on well-level therapeutic sensitivity. To characterize individual oligoclonal units of therapeutic response, we introduce a low-volume screening assay, including an automated alignment algorithm. The oligoclonal growth response was compared against validated markers of response, including well-level viability and markers of single-cell viability. Line-specific sensitivities were compared with clinical outcomes. Automated alignment algorithms were generated to match organoids across time using coordinates across a single projection of Z-stacked images. After screening for baseline size (50 μm) and circularity (>0.4), the match efficiency was found to be optimized by accepting the diffusion thresholded with the root mean standard deviation of 75 μm. Validated well-level viability showed a limited correlation with the mean organoid size (R = 0.408), and a normalized growth assayed by normalized changes in area (R = 0.474) and area (R = 0.486). Subclonal populations were defined by both residual growth and the failure to induce apoptosis and necrosis. For a culture with clinical resistance to gemcitabine and nab-paclitaxel, while a therapeutic challenge induced a robust effect in inhibiting cell growth (GΔ = 1.53), residual oligoclonal populations were able to limit the effect on the ability to induce apoptosis (GΔ = 0.52) and cell necrosis (GΔ = 1.07). Bioengineered approaches are feasible to capture oligoclonal heterogeneity in organotypic cultures, integrating ongoing efforts for utilizing organoids across cancer types as integral biomarkers and in novel therapeutic development.

Background: Therapeutic development in pancreatic ductal adenocarcinoma (PDAC) requires accurate models for assessing response. Patient-derived cancer organoids (PCOs) have been shown to faithfully recapitulate genomic features of advanced PDAC. Mechanisms of subclonal resistance or intratumoral heterogeneity cannot be characterized using well-level viability assays. Methods: A high-content imaging platform was adapted in low volume, 96-well format for using Cytation 5 (Biotek). PDAC PCOs were plated from organoid suspension in 1:1 reduced growth factor Cultrex plating 10uL per well. After 24h, physiologic chemotherapy was applied to match pharmacologic exposure of FOLFIRINOX (FFX) and gemcitabine/nab-paclitaxel (g/n-pac). Initial response was assessed by normalized change in PCO area at 72h. Subsequent viability staining was performed for single organoid level response for apoptosis (caspase-3/7-FITC) and necrosis (TO-PRO-3). Analyses were automated in Gen5.12 (Biotek) with selection for elements using circularity (>0.4) and alignment by location within 50% which represented 17.8% of g/npac and 18.5% of FFX treated PCOs. Individual organoids with mean area staining positive for apoptosis was increased in g/npac +67.3% (GD=2.96) and FFX +65.1 (GD=2.84) versus background control +12.8%. Individual organoids with mean with area staining positive for necrosis was increased in g/npac +47.3% (GD=1.88) as compared to both FFX +34.9 (GD=1.20, pConclusions: High content imaging in PDAC PCOs provide scalable model for response assessment. This method can track subclonal populations with residual growth after treatment with chemotherapy. Here, we provide proof of concept in patient-derived tissue for clinically relevant response assessment with increased induced necrosis with g/npac in comparison to FFX. Ongoing investigations are generating sensitivity thresholds to define subclonal populations with residual growth and viability for application in clinical prediction. Citation Format: Md Shahadat Hossan, Ethan S. Lin, Ellie E. Riedl, Austin Stram, Jamie M. Warner, Jeremy D. Kratz. Multiplexed therapeutic sensitivity of patient derived pancreatic cancer organoids after neoadjuvant chemotherapy by high content imaging [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A073.