Publications

Abstract Patient-Derived Organoids (PDO) and Xenografts (PDX) are the current gold standards for patient derived models of cancer (PDMC). Nevertheless, how patient tumor cells evolve in these models and the impact on drug response remains unclear. Herein, we compared the transcriptomic and chromatin accessibility landscapes of six matched sets of colorectal cancer (CRC) PDO, PDX, PDO-derived PDX (PDOX), and original patient tumors (PT) and discovered two major remodeling axes. The first axis delineates PDX and PDO from PT, and the second axis distinguishes PDX and PDO. PDOX were more similar to PDX than they were to PDO, indicating that the growth environment is a driving force for chromatin adaptation. Using bivariate genomic footprinting analysis, we identified transcription factors (TF) that differentially bind to open chromatins between matched PDO and PDOX. Among them, KLF14 and EGR2 footprints were enriched in all six PDOX relative to matched PDO, and silencing of KLF14 or EGR2 promoted tumor growth. Furthermore, EPHA4, a shared downstream target gene of KLF14 and EGR2, altered tumor sensitivity to MEK inhibitor treatment. Altogether, patient-derived CRC cells undergo both common and distinct chromatin remodeling in PDO and PDX/PDOX, driven largely by their respective microenvironments, which results in differences in growth and drug sensitivity and needs to be taken into consideration when interpreting their ability to predict clinical outcome.

Abstract Fibrolamellar Hepatocellular Carcinoma (FLC) is a usually lethal liver cancer affecting adolescents and young adults without previous liver disease. FLC is characterized by a ~400kb nucleotide deletion resulting in a fusion of a heatshock protein cofactor, DNAJB1, with the catalytic subunit of protein kinase A (PRKACA). One limitation to the study of this disease is the lack of in vitro models. Organoids have shown utility as a model system for studying many diseases, including cancer. This study describes the development and characterization of FLC tumor organoids. With IRB approval, normal liver and FLC tumor tissue was obtained from patients undergoing surgical resection. Liver cells were isolated from the tissue samples and placed into a 3D basement membrane matrix and the media supplemented with growth factors specific to promote liver organoid expansion. Normal and tumor organoids were analyzed by microscopy for morphology, by PCR for presence of the DNAJB1-PRKACA chimeric transcript, by Western blot for chimeric protein expression, and by RNA sequencing for transcriptomic changes. Eight organoid lines have been developed from normal liver and eight from FLC tumor tissue, including multiple independent organoids developed from different metastases from the same patient. A distinct morphological difference between normal liver organoids and FLC tumor organoids can be seen with either brightfield microscopy or with H&E. The DNAJB1-PRKACA chimeric transcript was detected in all of the tumor tissue and tumor organoids by RT-PCR and not detected in any of the normal tissue or organoids. The presence of the DNAJB1-PRKACA fusion protein was verified by Western blot in tumor organoids and tissue but not detectable in the normal tissue. The RNA sequencing analysis shows clustering of tumor organoids with tumor tissue. Tumor organoids injected subcutaneously in mice grew as tumors. Now that the normal and tumor organoids have been developed and validated, they are being used for screening for potential therapeutics. Citation Format: Nicole J. Croteau, David Requena, Gadi Lalazar, Denise Ng, Solomon Levin, Bassem Shebl, Ruisi Wang, William J. Hammond, James A. Saltsman, Helmuth Gehart, Hans Clevers, Michael P. LaQuaglia, Sanford Simon. Human liver organoids for disease modeling of fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3905.

<div>AbstractPurpose:<p>Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly extend tumor response in patients with metastatic estrogen receptor–positive (ER<sup>+</sup>) breast cancer, relapse is almost inevitable. This may, in part, reflect the failure of CDK4/6 inhibitors to induce apoptotic cell death. We therefore evaluated combination therapy with ABT-199 (venetoclax), a potent and selective BCL2 inhibitor.</p>Experimental Design:<p>BCL2 family member expression was assessed following treatment with endocrine therapy and the CDK4/6 inhibitor palbociclib. Functional assays were used to determine the impact of adding ABT-199 to fulvestrant and palbociclib in ER<sup>+</sup> breast cancer cell lines, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. A syngeneic ER<sup>+</sup> mouse mammary tumor model was used to study the effect of combination therapy on the immune system.</p>Results:<p>Triple therapy was well tolerated and produced a superior and more durable tumor response compared with single or doublet therapy. This was associated with marked apoptosis, including of senescent cells, indicative of senolysis. Unexpectedly, ABT-199 resulted in Rb dephosphorylation and reduced G<sub>1</sub>–S cyclins, most notably at high doses, thereby intensifying the fulvestrant/palbociclib–induced cell-cycle arrest. Interestingly, a CRISPR/Cas9 screen suggested that ABT-199 could mitigate loss of Rb (and potentially other mechanisms of acquired resistance) to palbociclib. ABT-199 did not abrogate the favorable immunomodulatory effects of palbociclib in a syngeneic ER<sup>+</sup> mammary tumor model and extended tumor response when combined with anti-PD1 therapy.</p>Conclusions:<p>This study illustrates the potential for targeting BCL2 in combination with CDK4/6 inhibitors and supports investigation of combination therapy in ER<sup>+</sup> breast cancer.</p></div>

Abstract Purpose There remains an unmet need for preclinical models to enable personalized therapy for ovarian cancer (OC) patients. Recently, patient-derived organoid (PDO) cultures of patients with OC have been established that faithfully represent the histopathological features and genomic landscape of the patient tumor. In this study, we evaluate the capacity of OC PDOs to predict clinical drug response and functional consequences of tumor heterogeneity. Experimental design 36 genomically characterized PDOs from 23 patients with known clinical histories were exposed to chemotherapeutics and targeted drugs. Results OC PDOs maintained genomic features of the original tumor lesion and recapitulated patient response to neoadjuvant carboplatin and paclitaxel combination treatment, according to distinct clinical outcomes (histopathological, biochemical and radiological). PDOs displayed inter-as well as intrapatient drug response heterogeneity, which could in part be explained by genetic aberrations. All PDOs were resistant to PARP-inhibitors, in accordance with homologous recombination pathway fidelity and genome-wide mutation context. KRAS, BRAF and NRAS mutation status predicted response to BRAF-inhibitor vemurafenib and pan-HER-inhibitor afatinib, and explained differential response among four PDOs derived from distinct tumor locations of an individual patient. Importantly, PDO drug screening identified sensitivity to at least one drug for the majority of patients (88%). Conclusions OC PDOs are a valuable preclinical model system that can provide insights in drug response for individual patients with OC, complementary to genetic testing. Generating PDOs of multiple tumor locations can improve clinical decision making and increase our knowledge on genetic and drug response heterogeneity.

Multi-omics profiling of colorectal cancer (CRC) patients and associated patient-derived organoids. Tumor organoids were characterized in steady-state and perturbed using Kinase inhibitors.

The epithelial-to-mesenchymal transition (EMT) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype. Here, we show that KSR1 and ERK promote EMT-like phenotype through the preferential translation of Epithelial-Stromal Interaction 1 (EPSTI1), which is required to induce the switch from E- to N-cadherin and coordinate migratory and invasive behavior. EPSTI1 is overexpressed in human colorectal cancer (CRC) cells. Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro, and reverses EMT-like phenotype, in part, by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression, ZEB1 and Slug. In CRC cells lacking KSR1, ectopic EPSTI1 expression restored the E- to N-cadherin switch, migration, invasion, and anchorage-independent growth. KSR1-dependent induction of EMT-like phenotype via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior.

Metastases undergo reconstruction Cancer cells from primary tumors can migrate to regional lymph nodes and distant organs. The prevailing model in oncology is that lymph node metastases give rise to distant metastases. This “sequential progression model” is the rationale for surgical removal of tumor-draining lymph nodes. Naxerova et al. used phylogenetic methods to reconstruct the evolutionary relationship of primary tumors, lymph node metastases, and distant metastases in 17 patients with colorectal cancer (see the Perspective by Markowitz). The sequential progression model applied to only one-third of the patients. In the other two-thirds, distant metastases and lymph node metastases originated from independent subclones within the primary tumor. Science , this issue p. 55 ; see also p. 35