Pancreatic Patient-derived Xenograft Research Articles

Abstract LB-A16: Targeting the WNT-pathway in 3D-cultured in vitro PDX models for pancreas cancer

Abstract Creating patient-derived xenograft (PDX) models from human cancers allows bio-banking, propagation, characterization and drug testing on a large panel of tumor variants. To scale up the number of compound tests, identify relevant compound doses, increase the throughput, and study relevant cancer-driving pathways we developed 3D in vitro culture methods for PDX model-derived cell isolates. In this effort, each individual model is subjected to a wide panel of culture medium formulations, amongst which some media contain WNT-pathway stimulating components. In one of our campaigns we identified an absolute dependency of one of the tested Pancreatic in vitro PDX models to the WNT-signaling potentiating factor R-Spondin, while other Pancreas models showed little or no dependency. To further study the dependency of Pancreatic PDX models on the functionality and signaling of the WNT-pathway, we exposed these in vitro PDX models to increasing doses of bio-similars of 18R5 (anti-FZD7), 54F28 (FZD8-Fc decoy receptor) and to the Porcupine inhibitor LGK974, which blocks WNT palmitoylation and therefore secretion. Our findings are that the WNT-pathway is functional and contributing to Pancreas cancer outgrowth and morphological differentiation at different extents and levels in different patient-models. Our in vitro PDX culture system in combination with 3D phenotypic analysis allows in-detail study of the WNT-pathway, as well as by supplementation or omission of secreted factors, addition of small molecule inhibitors and by exposure to antibodies. These in vitro PDX models can be used to test novel compounds and antibodies targeting the WNT pathway. Citation Format: Thomas Dijkmans. Targeting the WNT-pathway in 3D-cultured in vitro PDX models for pancreas cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A16.

Abstract LB-A18: Targeting the WNT-pathway in 3D-cultured in vitro PDX models for pancreas cancer

Abstract Creating patient-derived xenograft (PDX) models from human cancers allows bio-banking, propagation, characterization and drug testing on a large panel of tumor variants. To scale up the number of compound tests, identify relevant compound doses, increase the throughput, and study relevant cancer-driving pathways we developed 3D in vitro culture methods for PDX model-derived cell isolates. In this effort, each individual model is subjected to a wide panel of culture medium formulations, amongst which some media contain WNT-pathway stimulating components. We identified an absolute dependency of a pancreatic in vitro PDX tumor to the WNT-signaling potentiating factor R-Spondin, while other pancreas tumors showed little or no dependency. To further study the dependency of pancreatic PDX tumors on the functionality and signaling of the WNT-pathway, we exposed in vitro PDX cultures to increasing doses of anti-FZD7, anti-FZD8 and to the Porcupine inhibitor LGK974, which blocks WNT palmitoylation and therefore secretion. Our findings are that the WNT-pathway is functional and contributing to pancreas cancer outgrowth and morphological differentiation to different extents in different patient-models. Our in vitro PDX culture system in combination with 3D phenotypic analysis allows in-detail study of the WNT-pathway, as well as by supplementation or omission of secreted factors, addition of small molecule inhibitors and by exposure to antibodies. These in vitro PDX models can be used to test novel compounds and antibodies targeting the WNT-pathway and underscore the WNT-pathway as a potential target in pancreatic cancer. Citation Format: Thomas Dijkmans, Bram Herpers, Sander Basten, Kuan Yan, Torsten Giesemann, Julia Schueler, Leo Price. Targeting the WNT-pathway in 3D-cultured in vitro PDX models for pancreas cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A18.

Abstract 5112: Evaluation of FF-10502-01, a new pyrimidine nucleoside analogue, in pancreatic (PANC) patient-derived xenograft (PDX) models compared to gemcitabine and in combination with nab-paclitaxel

Abstract Introduction FF-10502-01 is a synthetic pyrimidine nucleoside analogue structurally similar to gem with a substitution of sulfur for oxygen in the pentose sugar. In previous studies, FF-10502-01 demonstrated preclinical efficacy across multiple solid tumors, including PANC cancer. In this study, we investigated the anti-tumor effect of FF-10502-01 in PANC PDX models. Methods 10 PANC PDX tumors were sourced from primary (2) or metastatic sites (8). 7 demonstrated high resistance (HR) to gem, 1 intermediate, and 2 low. In the dose-finding study, 3 PDX models were studied in 9 grps of NOD-SCID mice (n=10/grp), treated with 240 or 480 mg/kg FF-10502-01, 3 or 6 mg/kg nab-pac or 240 mg/kg gem, alone and in combination for 28d, followed by 28d observation. The definitive study consisted of 7 PDX models. 6 grps (n=10/grp) were treated with 240 or 480 mg/kg FF-10502-01, 6 mg/kg nab-pac or 240 mg/kg gem, alone and/or in combination for 28d, followed by 28d observation. After subcutaneous transplantation, animals were left undisturbed for 7d. Animals were monitored weekly and tumor volume measured with calipers. Average tumor volume (mm3) for each group at randomization into treatment grps ranged from 184.34 – 199.51 (SD ± 20.94 – 30.40). Statistical significance was determined using one-way ANOVA and Tukey’s test. Results At clinically relevant doses, FF-10502-01, alone and in combination with nab-pac demonstrated greater tumor growth suppression than vehicle-treated animals (p≤0.0001). In 7 models, FF-10502-01/nab-pac demonstrated higher tumor growth suppression than gem/nab-pac (p≤0.5, p≤0.01 or p≤0.001), irrespective of resistance to gem. In 3 models, there was no difference, but these models were highly responsive to gem/nab-pac, thus minimizing the effects of FF-10502-01/nab-pac. Despite statistical insignificance, FF-10502-01/nab-pac still demonstrated greater tumor growth inhibitory activity to gem/nab-pac. In HR gem models, FF-10502-01 was superior to gem (p≤0.0001, p≤0.001, p≤0.05) in 3 of 7, and FF-10502-01/nab-pac was superior to gem/nab-pac in 6 of 7 (p nab-pac (p≤0.0001, p≤0.0001, p≤0.01, p≤0.05). FF-10502-01/nab-pac also was more tolerable than gemcitabine/nab-pac, as demonstrated by less weight loss. Conclusions FF-10502-01 is a new pyrimidine nucleoside analogue with demonstrated preclinical efficacy in solid tumors, including PANC cancer. In PANC PDX models, FF-10502-01 alone and in combination with nab-pac demonstrated higher efficacy and better tolerability than gem alone or gem/nab-pac. FF-10502-01 is in Phase 1 clinical development. Citation Format: Takeaki Suzuki, Linda J. Paradiso, Jill Ricono, Jonathan Nakashima, Yoshihide Iwaki, Shinji Mima, Takayuki Yamada, Chihaya Kakinuma, Hiroyuki Iwamura, Shinichi Watanabe. Evaluation of FF-10502-01, a new pyrimidine nucleoside analogue, in pancreatic (PANC) patient-derived xenograft (PDX) models compared to gemcitabine and in combination with nab-paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5112. doi:10.1158/1538-7445.AM2017-5112

Abstract 3889: Novel drug candidates cep1430 and cep1507 for targeting pancreatic patient-derived xenograft cancer stem cell and circulating tumor cell models

Abstract Pancreatic cancer is the fourth leading cause of cancer mortality in the US, despite significant improvements in diagnostic imaging and operative mortality rates. The 5-year survival rate remains less than 6% because of microscopic or gross metastatic disease at time of diagnosis. Although the treatment of pancreatic cancer remains a challenge, it is entering new era with development of new strategies and trial designs. Because there is an increasing number of novel therapeutic agents and potential combinations available to test in patients with pancreatic cancer and in their PDX models, the identification of robust prognostic and predictive markers for Cancer Stem Cells (CSC) PDX and Circulating Tumor Cells (CTC) PDX and of new targets and relevant pathways is a top priority as well as the design of adequate trials incorporating molecular-driven hypothesis. Here, we examined the efficacy of combined treatments of CEP1430 and CEP1507 in human pancreatic patient-derived xenograft (PDX) cancer cells, and pancreatic PDX CSCs models from the same donors. CEP1430 inhibited the growth of CSCs and CEP1507 inhibited growth of CTCs, while gemcitabine suppressed the viability of non-CSCs [70%] (differentiated tumor Cells). Consistently, in vivo studies showed that CEP1507 when combined with gemcitabine could eliminate the engraftment of human pancreatic cancer CTCs [85%(n=50,p<0.001) and CEP1430 with gemcitabine selectively inhibited [80%] CSCs(n=50,p<0.001), more effectively than the individual agents. These data indicated that administration of CEP1430, which targets CSCs and CEP1507 targets CTCs, may constitute a potential therapeutic strategy for improving the efficacy of gemcitabine to eradicate advanced pancreatic cancer. This study has indicated the potential molecular therapeutic targets to eradicate the tumor – and metastasis-initiating cells (CTCs) and their progenies and development of new effective combination therapies against locally advanced and metastatic pancreatic cancer. Using PDX models, we confirmed the effectiveness and selectivity of the identified treatment responses with TGI at 97% with CEP1430. Note: This abstract was not presented at the meeting. Citation Format: Michael Sharma, Maulik Jain, Cristian Sharma, Karina Amezcua, Natalee Amezcua, Reed Hasson, Miriam Navel, Donna Stanton, Satya Narayan, Karl Kramberger, Rubio Punzalan, Jitesh Jani, Douglas Foster, Jay P. Sharma. Novel drug candidates cep1430 and cep1507 for targeting pancreatic patient-derived xenograft cancer stem cell and circulating tumor cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3889. doi:10.1158/1538-7445.AM2017-3889

Abstract 3073: Potential predictive biomarkers of clinical responses for a novel CDC7-selective inhibitor TAK-931

Abstract Cell division cycle 7 (CDC7) is a serine/threonine kinase, which plays important roles in initiation of DNA replication by phosphorylating MCM2. Kinase activity of CDC7 is controlled by its binding protein DBF4 in a cell-cycle dependent manner. Here we developed a potent CDC7 inhibitor TAK-931 (IC50< 0.3 nM) as a cancer therapeutic drug candidate, which exhibits a time-dependent ATP-competitive kinetics to its ATP-binding pocket. The selectivity studies using the 308 kinases revealed >120-fold selectivity of TAK-931 for CDC7 kinase inhibition compared to other kinase inhibitions. Treatment with TAK-931 suppressed the cellular MCM2 phosphorylation at Ser40, resulting in a delayed S phase progression, checkpoint activation, apoptosis, and potent growth suppression in various cancer cell lines. Furthermore, oral administration of TAK-931 as a single agent caused a significant antitumor activity in multiple xenograft models which include both cell line-based xenografts and patient-derived xenograft (PDX) models. These results demonstrate that TAK-931 is a highly potent and selective inhibitor of CDC7 kinase, and causes a potent antiproliferation both in vitro and in vivo studies using various cancer cells. Next, to identify potential predictive biomarkers to guide patient-selection strategies, in vitro cell panel screening of TAK-931 using was tested for its ability to antiproliferation in 246 cell lines, which includes both solid and hematological cancer cells. TAK-931 inhibited proliferation of multiple cancer cell lines, with mean concentration producing a half-maximal response (EC50) values ranging from 29.1 nM to > 30 μM (median = 554.5 nM). While the wide range of TAK-931 antiproliferative spectrum was observed, neither doubling speed nor CDC7 expression profile did predict tTAK-931 sensitivity in cancer cell lines. A correlative study of the tumor genetic mutations in relation to antiproliferative activity that KRAS mutant cancer cells were more sensitive to TAK-931 compared to KRAS non-mutant cell lines (p<0.05). We also confirmed this KRAS-associated antiproliferative effect of TAK-931 in the SW48 isogenic cell lines of KRAS mutations (G12V, G13D). The ectopic expressions of G12V- and G13D-KRAS mutations increased the TAK-931 sensitivity compared to the KRAS-wild parental SW48 cell line. Given that KRAS mutations are frequently detected in clinical pancreatic tumors, we next conducted in vivo efficacy studies using pancreatic PDX models. Consistent to our hypothesis from in vitro studies, KRAS-mutant pancreatic PDX tumors were more sensitive to TAK-931 than the KRAS-wild pancreatic tumors; all KRAS-mutant pancreatic PDX models we tested exhibited >60% TGI. Our findings suggest that the KRAS-mutant pancreatic tumors could be the potential candidate for the TAK-931 target indication. Citation Format: Kenichi Iwai, Tadahiro Nambu, Osamu Kurasawa, Noriko Uchiyama, Ryo Dairiki, Yukiko Yamamoto, Satoru Nishizawa, Mengkun Zhang, Yuko Ishii, Huifeng Niu, Akihiro Ohashi. Potential predictive biomarkers of clinical responses for a novel CDC7-selective inhibitor TAK-931 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3073. doi:10.1158/1538-7445.AM2017-3073

Abstract 3857: Patient-derived tumor xenografts as pharmacological model of human pancreatic ductal adenocarcinoma

Abstract INTRODUCTION and AIM Pancreatic ductal adenocarcinoma is a lethal disease with an overall survival rate of 5% and a median survival time of less than 6 months. Current systemic treatments offer only a modest benefit in symptom control and survival, thus surgery remains the most effective treatment. The exploitation of new therapeutics is an urgent need, but the research is braked by the limited value of the current preclinical models as predictors of patients response. The aim of our study is to develop patient-derived primary pancreatic ductal adenocarcinoma xenografts (PDAC-PDX) growing into the pancreas of recipient mice that retain features of the patient’s tumors and mimic the biological heterogeneity of human pancreatic cancer. METHODS Cancer specimens from patients undergoing cephalic duodenopancreatectomy were transplanted in NSG mice and orthotopically propagated by their re-implantation into the pancreas of SCID mice. Tumor burden was measured by abdominal palpation and by non invasive magnetic resonance imaging (MRI). PDAC-PDXs were characterized histologically, for mutational status (KRAS, TP53 and SMAD4), and responsiveness to gemcitabine. Gemcitabine was administered alone or combined with nab-paclitaxel, to mice bearing tumor in their pancreas; drugs were given intravenously on days 1 and 8 of each 21-day cycle to copy as much as possible patient’s recommendations. To determine the antitumor effects of the treatments, changes in tumor growth over time were evaluated by MRI and plasma level of Ca19-9. RESULTS Surgically resectable pancreatic adenocarcinoma (histologically confirmed) engrafted successfully in 5 cases (out of 11) and retained the molecular, morphological and biological features of the original patient tumors. Mutational landscape of the PDAC-PDXs reflect the clinical scenario: 80% are mutated in KRAS (G12D, G12R), 80% carry a TP53 mutation (I162F, R213*, Y220C, R248Q) and 20% show the deletion of SMAD4. Histological features recapitulate the human disease, the PDAC-PDXs exhibit a robust amount of host derived stroma with collagen deposition, constant through generations (up to five). Orthotopic growing PDAC-PDXs were marginal responsive to gemcitabine treatment, while significant response (stable disease and regression) was observed with the gemcitabine and nab-paclitaxel combination regimen, which was more efficacious than paclitaxel (equimolar dose). CONCLUSION PDAC-PDXs growing orthotopically in the pancreas of mice provide a valuable tool for the preclinical exploitation of novel therapeutics for pancreatic cancer. . Supported by Associazione Italiana per la Ricerca sul Cancro (AIRC 5 per mille n. 12182) and Fondazione “Eugenio Morandi” ONLUS per lo studio e la cura dei tumori del pancreas. Citation Format: Alessia Anastasia, Andrea Resovi, Carmen Ghilardi, Roberta Avigni, Edoardo Micotti, Lucia Minoli, Eugenio Scanziani, Carlo Corbellini, Eugenio Morandi, Dorina Belotti, Raffaella Giavazzi, MariaRosa Bani. Patient-derived tumor xenografts as pharmacological model of human pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3857. doi:10.1158/1538-7445.AM2017-3857

Abstract 4396: Multiomics assessment of the cancer and stromal compartments of patient-derived pancreatic xenografts reveals clinically-relevant subtypes and novel targeted therapies

Abstract Patient-derived xenografts (PDX) are appearing as a prime approach for preclinical studies despite being insufficiently characterized as a model of the human disease and its diversity. In this work, 29 PDX were obtained from either surgery or endoscopic ultrasound-guided fine needle aspirate of pancreatic adenocarcinoma. The extensive genomic profiling of these pancreatic PDX, revealed two clinically-relevant subtypes having broad similarities with human primary tumors. These subtypes are defined by highly specific DNA methylation and transcriptomic profiles (mRNA, miRNA or lncRNA) but are not distinguishable by exonic mutations or copy number aberrations. Moreover, by specifically analyzing the stroma transcriptome, as defined by the expression of murine transcripts, we found that it is able to stratify the patients with the same efficiency than the analysis of grafted human tumor cells. This finding suggest that transformed pancreatic cells drive the composition of their own stroma. Finally, the multiomics analysis pinpoints novel therapeutic targets, one of which we demonstrate to be an efficient method for treating pancreatic cancer. Overall, we show that PDX are trustworthy pre-clinical models of pancreatic adenocarcinoma including of unresectable tumors. Their multiomics profiling allow the independent analysis of the uncontaminated cancer or stromal compartments and discloses several original therapeutics targets. Citation Format: Remy Nicolle, Yuna Blum, Laetitia Marisa, Celine Loncle, Odile Gayet, Vincent Moutardier, Olivier Turrini, Marc Giovannini, Benjamin Bian, Martin Bigonnet, Marion Rubis, Nabila Elarouci, Lucile Armenoult, Mira Ayadi, Pauline Duconseil, Mohamed Gasmi, Mehdi Ouaissi, Aurélie Maignan, Gwen Lomberk, Jean-Marie Boher, Jacques Ewald, Erwan Bories, Jonathan Garnier, Anthony Goncalves, Flora Poizat, Jean-Luc Raoul, Veronique Secq, Stephane Garcia, Philippe Grandval, Marine Barraud-Blanc, Emmanuelle Norguet, Marine Gilabert, Jean-Robert Delpero, Julie Roques, Ezequiel Calvo, Fabienne Guillaumond, Sophie Vasseur, Raul Urrutia, Aurélien de Reyniès, Nelson Dusetti, Juan Iovanna. Multiomics assessment of the cancer and stromal compartments of patient-derived pancreatic xenografts reveals clinically-relevant subtypes and novel targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4396. doi:10.1158/1538-7445.AM2017-4396

Abstract IA21: Targeting WEE1 kinase to potentiate chemoradiation in the treatment of pancreatic cancer

Abstract Pancreas cancer is projected to cause approximately 42,000 deaths in 2016, making it the third greatest cause of cancer mortality (behind lung and colorectal, but greater even than breast). Systemic failure remains the dominant cause of death, although local failure is responsible for up to 1/3 of the pancreatic cancer related mortality. We have focused on improving the treatment of locally advanced pancreatic cancer, defined as disease which cannot be resected but has not overtly metastasized, where the issue of local control becomes even more important, and have hypothesized that the treatment of locally advanced pancreatic cancer will require control of both the gross primary tumor as well as the microscopic disease that exists in virtually every patient. Gemcitabine has been a mainstay of treatment for pancreatic cancer, and we have standardized combining it with radiation for locally advanced disease. Because our initial studies revealed that activation of the DNA damage response, particularly activation of CHK1 and WEE1, were crucial in the defense pancreatic cancers mount against chemotherapy and radiation, we have studied the effect of CHK1 and WEE1 inhibitors on gemcitabine and gemcitabine-radiation mediated cytotoxicity. These inhibitors abrogate the G2 checkpoint after DNA damage. The initial hypothesis for using CHK1 and WEE1 inhibitors was that cancer cells lacking a p53 checkpoint would be selectively sensitized, compared to normal cells, because normal cells would be protected by an intact G1 checkpoint. After initial studies with several agents, we have focused on the WEE1 inhibitor AZD1775, chiefly because it was available for a clinical trial. We have carried out both mechanistic and clinical studies using AZD1775. Mechanistically, we have evaluated both the contributions of homologous recombination repair (HRR) inhibition and G2 checkpoint abrogation by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistoneH3 flow cytometry, respectively in pancreatic cancer model systems. AZD1775 causes inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. Sensitization by AZD1775 is associated with persistent γH2AX and inhibition of RAD51 focus formation. Using BRCA2 isogenic cells, we have found that AZD1775 sensitizes to gemcitabine-radiation in BRCA2 wild type cells, while in BRCA2 null cells AZD1775 did not sensitize despite significant G2 checkpoint abrogation. In vivo, in patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibits tumor growth and impairs RAD51 focus formation in response to gemcitabine-radiation. We've concluded that, whereas sensitization is accompanied by inhibition of CDK1 phosphorylation and abrogation of the G2 checkpoint, this mechanism is not sufficient for sensitization. Our findings suggest that sensitization to chemoradiation by WEE1 inhibition also requires inhibition of HRR. The importance of HRR has also supported studies combining CHK1 or WEE1 inhibition with a PARP inhibitor, which would be anticipated to potentiate the effects of HRR inhibition. Based on these studies, we have launched a clinical trial combining gemcitabine and gemcitabine-radiation, at standard doses, with dose escalating AZD1775 for patients with locally advanced pancreatic cancer. Patients receive one cycle of gemcitabine with AZD1775, then 2 cycles of gemcitabine-radiation with AZD1775, then another cycle of AZD1775-gemcitabine, followed by assessment for response and toxicity. Optional cycles of AZD1775 and gemcitabine are then given to patients who have not progressed. AZD1775 has been escalated according to a Time-to-Event Continual Reassessment Model, in which the dose for each patient is based on the toxicity profile of all prior patients, and the goal is to establish a tolerable dose of the combination therapy and to estimate the response and survival. With 21 patients entered onto the study, the median survival has not been reached, and the 64% survival at the 18-month landmark point exceeds the 18-month median survival that was the agreed on endpoint for triggering a larger trial. The trial also includes a pharmacodynamic endpoint of a hair follicle biopsy to assess target engagement. In summary, these findings suggest that WEE1 inhibition may act through both G2 checkpoint abrogation but, more importantly, inhibition of HRR, and is a promising potentiator of gemcitabine-radiation in the treatment of locally advanced pancreatic cancer. Supported by P50 CA130810, R01 CA163895, P30 CA46592, and the Taubman Institute Citation Format: Theodore S. Lawrence, Meredith A. Morgan. Targeting WEE1 kinase to potentiate chemoradiation in the treatment of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA21.

EGFR-Targeted Metal Chelating Polymers (MCPs) Harboring Multiple Pendant PEG2K Chains for MicroPET/CT Imaging of Patient-Derived Pancreatic Cancer Xenografts.

In this study, we developed a new generation of metal chelating polymer (MCP) reagents that carry multiple polyethylene glycol (PEG) pendant groups to provide stealth to MCP-based radioimmunoconjugates (RICs). We describe the MCP synthesis for covalent attachment to panitumumab F(ab')2 fragments (pmabF(ab')2) in which different numbers of pendant methoxy-PEG chains [M = 2000, ∼45 ethylene glycol (EG) repeat units, referred to as PEG2K] are incorporated into the polymer backbone. The pendant PEG2K chains were designed to provide a protein-repellant corona so that metal chelators attached closer to the polymer backbone will be less apparent to the physiological environment. DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) groups to chelate 64Cu were installed on these conjugates to be employed for PET imaging. The conjugation of MCPs to pmabF(ab')2 was based on a UV quantifiable bis-aromatic hydrazone formation under mild conditions (pH 5-6) between an aromatic aldehyde introduced on ε-NH2 groups of lysines in the F(ab')2 fragments and a hydrazinonicotinamide (HyNic) group installed on the initiating end of the MCP. Three MCPs with 17 polyglutamide (PGlu) repeat units, DOTA chelators and with an average of 2, 4, and 8 pendant PEG2K chains were studied to examine their in vitro and in vivo characteristics, as well as their potential for PET/CT imaging. A pmabF(ab')2-MCP conjugate carrying 2 PEG2K and one carrying 8 PEG2K pendant chains in the polymer were selected for microPET/CT imaging and biodistribution studies in tumor-bearing mice. Orthotopic pancreatic patient-derived xenografts tumors were visualized by PET/CT imaging. These RICs showed low levels of liver and spleen uptake along with even lower levels of kidney uptake. These encouraging results confirm the stealth properties of the MCPs with pendant PEG2K chains.

Abstract B72: Pancreatic cancer cell drives stroma composition

Abstract The extensive desmoplasia in pancreatic ductal adenocarcinoma (PDAC) has raised major interrogations on its role and function in the carcinogenic process. Patient-derived xenograft (PDX) offers an ideal setting to distinguish and to study the interactions between the cancerous epithelial cells and its stroma. Indeed, sequencing profiles of a mix of cancerous/Human and stroma/Mouse cells can be analyzed separately in silico by unambiguously assigning each sequence to the human or mouse genome. Using RNA sequencing, we profiled 30 pancreatic tumor PDXs and extracted the transcriptome profiles of epithelial cancerous cells as well as their corresponding stroma. On average, 70% of RNA sequencing reads were specifically attributed to a human origin, and therefore an epithelial origin, while 22% of RNA sequencing reads were mouse-specific. Using the rate of mouse sequences as a surrogate of the proportion of non-transformed cells, we observe a high variability in the infiltration level from 7% to 60%. The estimation was also consistent in whole exome sequencing (using the same sequencing process) and with the histological quantification of fibrotic tissue. By specifically analyzing the gene expression in mouse-stromal cells, we show that their transcriptomic profile is consistent with the recent description of human PDAC in situ tumors with high levels of genes of the reported activated-stroma and normal-stroma signatures. We also show that stromal cells over-express genes involved in the SLIT/ROBO axon guidance signalling pathway, in angiogenesis as well as a large number of collagens, cytokines and ligands associated with growth and developmental pathways. Recent studies identified two major subtypes of PDAC from transcriptomic analysis: a well differentiated, often referred as classical, and an undifferentiated, previously recognized as Basal, Quasi-Mesenchymal or Squamous. The stroma characterized in this work broadly reflects this heterogeneity with stromal gene expression signatures predictive of each subtype. For instance, collagens are significantly over-expressed in the stroma of squamous tumors. The concomitant analysis of transcriptomic profiles of both subtypes shows potential cross-talks between cancerous and stromal cells. Particularly in Squamous tumors, genes implicated in the axon-guidance and Wnt pathways are significantly upregulated in both, stroma and transformed cells. On the other hand, the stroma and transformed cells of Classical tumors shows an upregulation of complementary genes associated with several metabolic pathways. Taken together, our transcriptomic analysis reveals that human transformed pancreatic cells determine the composition, quantitatively and qualitatively, of stroma mouse cells in a PDX model. This model also reveals a broad variability of PDAC stromas and highlights potential cross-talks between them through known and novel pathways. Citation Format: Rémy Nicolle, Yuna Blum, Laetitia Marisa, Jonathan Garnier, Benjamin Bian, Celine Loncle, Martin Bigonnet, Odile Gayet, Vincent Moutardier, Pauline Duconseil, Mohamed Gasmi, Mehdi Ouaissi, Olivier Turrini, Marc Giovannini, Aurélie Maignan, Jean-Marie Boher, Jacques Ewald, Erwan Bories, Marc Barthet, Anthony Goncalves, Flora Poizat, Jean-Luc Raoul, Veronique Secq, Stephane Garcia, Philippe Grandval, Marine Barraud-Blanc, Emmanuelle Norguet, Marine Gilabert, Jean-Robert Delpero, Ezequiel Calvo, Aurélien de Reyniès, Juan Iovanna, Nelson Dusetti.{Authors}. Pancreatic cancer cell drives stroma composition. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B72.

Targeted delivery of chemotherapy using HSP90 inhibitor drug conjugates is highly active against pancreatic cancer models.

The lack of effective treatment modalities is a major problem in pancreatic cancer (PCa), a devastating malignancy that is nearly universally driven by the “undruggable” KRAS and TP53 cancer genes. Poor tumor tissue penetration is the major source of resistance in pancreatic cancer where chemotherapy is the mainstay of treatment. In this study we exploited the selective tumor-targeting properties of the heat shock 90 protein inhibitors as the vehicle for drug delivery to pancreatic tumor tissues. STA-12-8666 is a novel esterase-cleavable conjugate of an HSP90i and a topoisomerase I inhibitor, SN-38. STA-12-8666 selectively binds activated HSP90 and releases its cytotoxic payload resulting in drug accumulation in pancreatic cancer cells in vivo. We investigated the preclinical activity of STA-12-8666 in patient derived xenograft and genetic models of pancreatic cancer.Treatment with STA-12-8666 of the KPC mice (knock-in alleles of LSL-KrasG12D, Tp53fl/fl and Pdx1-Cre transgene) at the advanced stages of pancreatic tumors doubled their survival (49 days vs. 74 days, p=0.008). STA-12-8666 also demonstrated dramatically superior activity in comparison to equimolar doses of irinotecan against 5 patient-derived pancreatic adenocarcinoma xenografts with prolonged remissions in some tumors. Analysis of activity of STA-12-8666 against tumor tissues and matched cell lines demonstrated prolonged accumulation and release of cytotoxic payload in the tumor leading to DNA damage response and cell cycle arrest.Our results provide a proof-of-principle validation that HSP90i-based drug conjugates can overcome the notorious treatment resistance by utilizing the inherently high affinity of pancreatic cancer cells to HSP90 antagonists.

Generation and molecular characterization of pancreatic cancer patient-derived xenografts reveals their heterologous nature.

Pancreatic ductal adenocarcinoma (PDAC) is the most challenging type of cancer to treat, with a 5-year survival rate of <10%. Furthermore, because of the large portion of the inoperable cases, it is difficult to obtain specimens to study the biology of the tumors. Therefore, a patient-derived xenograft (PDX) model is an attractive option for preserving and expanding these tumors for translational research. Here we report the generation and characterization of 20 PDX models of PDAC. The success rate of the initial graft was 74% and most tumors were re-transplantable. Histological analysis of the PDXs and primary tumors revealed a conserved expression pattern of p53 and SMAD4; an exome single nucleotide polymorphism (SNP) array and Comprehensive Cancer Panel showed that PDXs retained over 94% of cancer-associated variants. In addition, Polyphen2 and the Sorting Intolerant from Tolerant (SIFT) prediction identified 623 variants among the functional SNPs, highlighting the heterologous nature of pancreatic PDXs; an analysis of 409 tumor suppressor genes and oncogenes in Comprehensive Cancer Panel revealed heterologous cancer gene mutation profiles for each PDX-primary tumor pair. Altogether, we expect these PDX models are a promising platform for screening novel therapeutic agents and diagnostic markers for the detection and eradication of PDAC.

Abstract A42: Using a PDX tumor bank to screen for cancer stem cell therapies

Abstract OncoMed Pharmaceuticals is focused on discovering novel therapies that target cancer stem cells (CSCs), specifically those which depend on the Notch or Wnt pathways. Since Patient Derived Xenografts (PDXs) recapitulate both tumor cell heterogeneity and maintain the histopathological characteristics of the original tumor, they represent an important preclinical model for CSC drug discovery and can be used to effectively screen for new therapeutic candidates. To this end, OncoMed has established a fully characterized PDX Tumor Bank for its monoclonal antibody discovery and developmental efforts and has used PDX models to advance seven candidates into clinical trials. The goal of this study is to: 1) review the operational steps needed to propagate PDX's; 2) define the quality controls necessary to maintain these tumors; 3) assess how the Tumor Bank has helped in the selection and advancement of these targeted biologics into the clinic; and 4) review the role of the Tumor Bank in defining biomarkers for clinical use. Tumor specimens were received from surgery and processed into fragments, cell clumps, or dissociated cells. Tumor tissue or cells were either implanted into NOG or Nod/scid mice or frozen for later implantation. Primary tumors that grew were serially transplanted to establish working stocks for drug screening experiments. Every tumor was characterized for a variety of molecular, cellular, and tumor endpoints. All pertinent data and samples were captured into a custom designed database which included lineage diagrams to easily track tumor propagation and characterization endpoints. The quality control of the established PDXs must be carefully monitored throughout the process. We have used DNA fingerprinting to ensure the identification of each tumor and subsequent passages, but the larger problem of identifying and monitoring the development of spontaneous lymphomas (both human and murine) that can infiltrate and contaminate the PDX required a rigorous monitoring strategy. The infection of mice with Lactate Dehydrogenase Elevating Virus from contaminated reagents can severely affect animal health and, therefore the screening process. Surprisingly, the misdiagnosis of tumors received from surgery was higher than expected and resulted in tumors being correctly reclassified before being used. The strategy employed to effectively screen selected targets in the Notch and Wnt pathways was to first review the molecular characterization data from our PDX models, and then select appropriate models for in vivo efficacy testing. In order to assess how the Tumor Bank has helped both in the screening process and the identification of biomarkers, we will review both pre-clinical and clinical data for selected OncoMed antibodies. As an example, one anti-CSC agent, OMP-59R5 (Tarextumab), which targets Notch2/3 was tested in ten pancreatic PDX models. Six pancreatic PDX tumors were responders to anti-NOTCH2/3 while four were non-responders. Bioinformatic analysis of the responder/non-responder data sets identified tumors that had high Notch3 gene expression as responders to OMP-59R5 treatment. Based on this preclinical data, Notch3 levels were evaluated in a Phase 1b pancreatic trial as a potential predictive biomarker. In this trial, higher response rate and longer survival was noted in patients with Notch3 high tumors receiving GEM/Nab-P/ Tarextumab (at 5-15mg/kg). These observations are being tested in a placebo-controlled, randomized Ph2 setting. The creation of a working PDX Tumor Bank across multiply human solid tumor types has allowed us to implement an effective preclinical screening program to select candidate biologics and potential predictive biomarkers for targets in the Notch and Wnt pathways. Citation Format: James Evans, Chun Zhang, Angie InKyung Park, Alayne Brunner, Min Wang, Cristina Dee-Hoskins, Roger Lopez, Xiaomei Song, Kellie Pickell, Wan-Cheng Yen, Marcus Fischer, Raymond Tam, Gilbert O'Young, Jakob Dupont, Lei Zhou, Austin Gurney, John Lewicki, Tim Hoey, Ann M. Kapoun, Belinda Cancilla. Using a PDX tumor bank to screen for cancer stem cell therapies. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A42.

Abstract 633: Gene-expression characterization of bioluminescent PDX in orthotopic and ectopic models of pancreatic cancer and metastatic triple-negative breast cancer

Abstract Background: Patient derived xenografts (PDX) are regarded as the gold-standard for translational preclinical cancer research due to their preservation of tumor heterogeneity and propagation in vivo. Similarly, grafting human tumor cells of a particular tissue origin into the corresponding orthotopic context enhances translational accuracy; with recapitulation of tumor::microenvironment biology, as well as more accurate tumor progression kinetics and metastasis, a s well as for systemic delivery of therapies as compared to traditional subcutaneous xenografts. Bioluminescent luciferase-reporters into traditional cancer cell lines has allowed for in-life imaging of tumor growth kinetics in real-time, within obfuscated internal organs, improving experimental precision. We have reported on the development and validation of bioluminescent pancreatic cancer and breast cancer PDX in clinically recapitulative orthotopic models. Here, we characterize these bioluminescent PDX by gene-expression profiling; comparing orthotopic and ectopically propagated bioluminescent PDX to their non-transduced parental PDX counterparts. Methods: Pancreatic (PANx-005-Luc) or metastatic breast (HBCx-14-Luc) PDXs stably transduced with lentiviral luciferase were dissociated and implanted orthotopically into pancreas or mammary fat pad respectively or subcutaneously into the flank of NOD scid gamma (NSG) mice. Likewise, the parental reporter-free PANx-005 or HBCx-14 PDX were implanted subcutaneously into NSG mice. For luciferase-expressing PDX implanted orthotopically, tumor growth was monitored in-life (Xenogen IVIS® Lumina Series III instrument (IVIS)). For the PANx-005-Luc, orthotopic tumors were harvested at a mean tumor radiance (TR) of 2.53 × 106 + 1.63 × 106 ph/s, while ectopic luciferase-transduced and parental tumors were harvested at a mean tumor volume of 530 + 337mm3. For the HBCx-14-Luc, orthotopic tumors were harvested individually when TR reached 6.5 × 109 ph/s while ectopic luciferase-transduced or parental tumors were harvested when tumor volumes reached 1500 + 362.5mm3. Tumor samples were fixed in 10% neutral buffered formalin and embedded in paraffin-blocks for histological analysis. RNA was extracted from all tumors and full-coverage human genome gene-expression profiling was performed. Major differences observed in microarray data was validated via quantitative biochemical assays. Results: Stable expression of lentiviral luciferase did not result in significant gene-expression changes to PDX lines, while site of implantation altered some gene-expression characteristics of the PDX. Conclusions: The introduction of an integrating stable bioluminescent reporter does not significantly alter the gene-expression characteristics of PANx-005 or HBCx-14 PDX. Site of implantation plays a role in gene-expression of PDX tumor models. Citation Format: Benjamin G. Cuiffo, Olivier Deas, Ingrid Rankin, Gregory D. Lyng, Stephano Cairo, Katie Pedrick, Alexandra Kury, Enora Le Ven, Jean-Gabriel Judde, Stephen T. Sonis. Gene-expression characterization of bioluminescent PDX in orthotopic and ectopic models of pancreatic cancer and metastatic triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 633.

Abstract 3129: Predictive biomarker identification for response to vantictumab (OMP-18R5; anti-Frizzled) using primary patient-derived human pancreatic tumor xenografts

Abstract Background: The WNT/ β-catenin signaling pathway has been shown to play a key role in both normal development and tumorigenesis (Polakis, 2007; MacDonald et al., 2009). We have developed a monoclonal antibody, vantictumab, that blocks canonical WNT/β-catenin signaling through binding of five FZD receptors (1, 2, 5, 7, 8). This antibody inhibits the growth of several tumor types, including pancreas, breast, colon and lung. Furthermore, our studies showed that vantictumab reduces tumor-initiating cell frequency and exhibits synergistic activity with standard-of-care (SOC) chemotherapeutic agents (Gurney et al., 2012). Material and methods: We set out to identify a predictive biomarker for the response to vantictumab in pancreatic cancer patients by analyzing mRNA-seq gene expression data from 14 patient-derived xenograft (PDX) models. These 14 minimally passaged pancreatic xenograft tumors were tested in vivo and their responses to vantictumab, in combination with the current SOC gemcitabine and nab-paclitaxel were established. Samples from these experiments were collected for Pharmacodynamic (PD) biomarker analysis. We utilized a two-sample Welch's t-test to identify genes that can distinguish between responders and non-responders and the K-nearest neighbor (KNN, Altman 1992) algorithm for classification. A leave-one-out cross-validation was used to measure area under the ROC curve (Fawcett et al., 2006, AUC), accuracy (ACC), positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of the model. Results: PD biomarker analysis confirmed inhibition of genes in Wnt and stem cell pathways by vantictumab in combination with gemcitabine as well as gemcitabine plus nab-paclitaxel. The selected 3-gene signature comprising TGFB3, IGF2 and SMO achieved the best performance (AUC = 0.875, ACC = 0.93, PPV = 0.91, NPV = 1, sensitivity = 1, specificity = 0.75) in the 14 PDX pancreatic tumor models. In addition, a strong correlation between the gene signature biomarker and the ratio of tumor inhibition (RTI) in the pancreatic xenograft experiments was observed. The identified 3-gene biomarker was used to predict the response to vantictumab in combination with gemcitabine and nab-paclitaxel in three additional pancreatic PDX tumor models. The efficacy in the three models was successfully predicted by the biomarker. Conclusions: The 3-gene biomarker is being evaluated in a Phase 1b study of vantictumab in combination with gemcitabine and nab-paclitaxel in previously untreated stage IV pancreatic cancer (NCT02005315). Citation Format: CHUN ZHANG, Fiore Cattaruzza, Pete Yeung, Wan-Ching Yen, Marcus Fischer, Claire Guo, Alayne Brunner, Min Wang, Belinda Cancilla, Austin Gurney, Rainer Brachmann, John Lewicki, Tim Hoey, Ann M. Kapoun. Predictive biomarker identification for response to vantictumab (OMP-18R5; anti-Frizzled) using primary patient-derived human pancreatic tumor xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3129.

Pancreatic cancer stem cells in patient pancreatic xenografts are sensitive to drozitumab, an agonistic antibody against DR5

BackgroundTherapeutic resistance and tumor recurrence are two major hurdles in the treatment of pancreatic ductal adenocarcinoma. Recent findings suggest that both of these attributes are associated with a small subset...

Targeting hypoxic microenvironment of pancreatic xenografts with the hypoxia-activated prodrug TH-302.

Previous reports have suggested that the hypoxic microenvironment provides a niche that supports tumor stem cells, and that this might explain clinical observations linking hypoxia to metastasis. To test this, we examined the effects of a hypoxia-activated prodrug, TH-302, on the tumor-initiating cell (TIC) frequency of patient-derived pancreatic xenografts (PDX).The frequencies of TIC, measured by limiting dilution assay, varied widely in 11 PDX models, and were correlated with rapid growth but not with the levels of hypoxia. Treatment with either TH-302 or ionizing radiation (IR), to target hypoxic and well-oxygenated regions, respectively, reduced TIC frequency, and the combination of TH-302 and IR was much more effective in all models tested. The combination was also more effective than TH-302 or IR alone controlling tumor growth, particularly treating the more rapidly-growing/hypoxic models. These findings support the clinical utility of hypoxia targeting in combination with radiotherapy to treat pancreatic cancers, but do not provide strong evidence for a hypoxic stem cell niche.

Abstract A9: Bioluminescent orthotopic PDX models of primary pancreatic cancer and residual/metastatic breast cancer predict efficacy of standard of care and experimental treatments

Abstract Background: Only 5% of investigational anti-cancer agents are ultimately clinically successful. This may be attributed in part to the historical preclinical use of human cell lines that poorly recapitulate tumor heterogeneity in imperfect xenograft models. To model critical influences of tissue environment on tumor behavior and therapeutic response, heterogeneous patient-derived xenografts (PDX) can be assayed following implantation at orthotopic sites in rodents. Challenges to this approach include inaccessibility of certain organs or inability to track residual or metastatic disease. To address these problems, we utilized pancreatic (PANx-005) or metastatic breast (HBCx-14) PDXs transduced with stable bioluminescent reporters in efficacy studies of standard of care (SOC) and experimental treatments in clinically recapitulative models. Methods: Freshly excised PDXs were transduced with lentiviral vectors stably expressing luciferase, and implanted orthotopically into NOD scid gamma (NSG) mice. For our primary pancreatic cancer model, the pancreas was surgically exposed, and ∼2.5 × 106 PANx-005-Luc cells were inoculated directly. Post-op, tumor growth was monitored in-life (Xenogen IVIS® Lumina Series III instrument (IVIS)). Mice were randomized into treatment groups when mean tumor radiance (TR) reached 3.0 × 106 photons/sec. Control animals received no treatment; SOC groups received either focused fractionated radiation (12 Gy as 3×4 Gy fractions, Q5D (SRT)) or gemcitabine (75 mg/kg, 2QWx4, i.p.), and the experimental group received (+)-JQ-1 BET bromodomain inhibitor (50 mg/kg, QD, i.p.). For the residual/metastatic breast cancer model, ∼1.5 × 106 HBCx-14-Luc were injected directly into the 4th inguinal mammary fat pad. Tumor growth and response were monitored throughout the study by IVIS. When individual TR reached 7.5 × 109 photons/sec, the primary tumor was resected and the animal enrolled into a treatment group to be treated for residual/metastatic disease. Control animals received no treatment; SOC groups received either SRT, capacetabine (540 mg/kg QDx5/ 1week rest, p.o.) or docetaxel (20 mg/kg, Q3Wx2, i.p.); while experimental animals received (+)-JQ-1 (50 mg/kg, QD, i.p.). Results: Tumor seeding approached 100% in both models. Growth kinetics resembled clinical indications, including rapid growth and lung metastases for the HBCx-14 tumors, and slower growth of PANCx-005 tumors. Efficacies of individual treatments recapitulated clinical responses: HBCx-14 tumors were high responders to capecitabine and (+)-JQ-1, and low-responders to docetaxel, while PANx-005 tumors displayed high response to (+)-JQ-1 and lower response to gemcitabine. Conclusions: The translational predictivity of preclinical cancer models is enhanced by the use of human PDXs that preserve tumor heterogeneity, are assayed at orthotopic sites, and are utilized in models that recapitulate clinical situations (e.g. treatment of residual disease vs primary tumor). The use of stable bioluminescent reporters in such assays greatly enhances precision in monitoring tumor growth and treatment response. Currently available preclinical oncology models are more recapitulative, precise and predictive than ever before, and appear poised to engender improved translational success. Citation Format: Benjamin G. Cuiffo, Olivier Deas, Gregory D. Lyng, Stephano Cairo, Enora Le Ven, Jean-Gabriel Judde, Stephen T. Sonis. Bioluminescent orthotopic PDX models of primary pancreatic cancer and residual/metastatic breast cancer predict efficacy of standard of care and experimental treatments. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A9.

Sensitization of Pancreatic Cancers to Gemcitabine Chemoradiation by WEE1 Kinase Inhibition Depends on Homologous Recombination Repair

To improve the efficacy of chemoradiation therapy for locally advanced pancreatic cancer and begin to establish patient selection criteria, we investigated the combination of the WEE1 inhibitor AZD1775 with gemcitabine-radiation in homologous recombination (HR) repair proficient and deficient pancreatic cancers. Sensitization to gemcitabine-radiation by AZD1775 was assessed in pancreatic cancer cells by clonogenic survival and in patient-derived xenografts by tumor growth. The contributions of HR repair inhibition and G2 checkpoint abrogation to sensitization were assessed by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistone H3 flow cytometry, respectively. We found that AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type but not BRCA2 mutant pancreatic cancer cells. In all cells, AZD1775 caused inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. However, sensitization by AZD1775 was associated with persistent γH2AX and inhibition of RAD51 focus formation. In HR-proficient (BRCA2 wild-type) or -deficient (BRAC2 null) isogenic cells, AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type, but not in BRCA2 null cells, despite significant G2 checkpoint abrogation. In patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibited tumor growth and impaired RAD51 focus formation in response to gemcitabine-radiation. In conclusion, WEE1 inhibition by AZD1775 is an effective strategy for sensitizing pancreatic cancers to gemcitabine chemoradiation. Although this sensitization is accompanied by inhibition of CDK1 phosphorylation and G2 checkpoint abrogation, this mechanism is not sufficient for sensitization. Our findings demonstrate that sensitization to chemoradiation by WEE1 inhibition results from inhibition of HR repair and suggest that patient tumors without underlying HR defects would benefit most from this therapy.

Gene Expression Ratios Lead to Accurate and Translatable Predictors of DR5 Agonism across Multiple Tumor Lineages.

Death Receptor 5 (DR5) agonists demonstrate anti-tumor activity in preclinical models but have yet to demonstrate robust clinical responses. A key limitation may be the lack of patient selection strategies to identify those most likely to respond to treatment. To overcome this limitation, we screened a DR5 agonist Nanobody across >600 cell lines representing 21 tumor lineages and assessed molecular features associated with response. High expression of DR5 and Casp8 were significantly associated with sensitivity, but their expression thresholds were difficult to translate due to low dynamic ranges. To address the translational challenge of establishing thresholds of gene expression, we developed a classifier based on ratios of genes that predicted response across lineages. The ratio classifier outperformed the DR5+Casp8 classifier, as well as standard approaches for feature selection and classification using genes, instead of ratios. This classifier was independently validated using 11 primary patient-derived pancreatic xenograft models showing perfect predictions as well as a striking linearity between prediction probability and anti-tumor response. A network analysis of the genes in the ratio classifier captured important biological relationships mediating drug response, specifically identifying key positive and negative regulators of DR5 mediated apoptosis, including DR5, CASP8, BID, cFLIP, XIAP and PEA15. Importantly, the ratio classifier shows translatability across gene expression platforms (from Affymetrix microarrays to RNA-seq) and across model systems (in vitro to in vivo). Our approach of using gene expression ratios presents a robust and novel method for constructing translatable biomarkers of compound response, which can also probe the underlying biology of treatment response.