Patient-derived Pancreatic Xenograft Models Research Articles

Prediction the clinical EPR effect of nanoparticles in patient-derived xenograft models.

Many preclinically tested nanoparticles in existing animal models fail to be directly translated into clinical applications because of their poor resemblance to human cancer. Herein, the enhanced permeation and retention (EPR) effect of glycol chitosan nanoparticles (CNPs) in different tumor microenvironments (TMEs) was compared using different pancreatic tumor models, including pancreatic cancer cell line (BxPC3), patient-derived cancer cell (PDC), and patient-derived xenograft (PDX) models. CNPs were intravenously injected into different tumor models, and their accumulation efficiency was evaluated using non-invasive near-infrared fluorescence (NIRF) imaging. In particular, differences in angiogenic vessel density, collagen matrix, and hyaluronic acid content in tumor tissues of the BxPC3, PDC, and PDX models greatly affected the tumor-targeting efficiency of CNPs. In addition, different PDX models were established using different tumor tissues of patients to predict the clinical EPR effect of CNPs in inter-patient TMEs, wherein the gene expression levels of PECAM1, COL4A1, and HAS1 in human tumor tissues were observed to be closely related to the EPR effect of CNPs in PDX models. The results suggested that the PDX models could mimic inter-patient TMEs with different blood vessel structures and extracellular matrix (ECM) content that critically affect the tumor-targeting ability of CNPs in different pancreatic PDX models. This study provides a better understanding of the heterogeneity and complexity of inter-patient TMEs that can predict the response of various nanoparticles in individual tumors for personalized cancer therapy.

Abstract 3931: TGN-1062 inhibits CDK7 and augments the effects of gemcitabine and nab-paclitaxel in pancreatic cancer models

Abstract Purpose: Overexpression of Cyclin-Dependent Kinase 7 (CDK7) is linked to poor clinical outcomes in a variety of cancer types including AML, breast, ovarian, pancreatic, and lung cancers. CDK7 plays a dual role in tumor progression by regulating the cell cycle via phosphorylation of members of the CDK family and stimulation of transcription via phosphorylation of RNA Polymerase II. CDK7 has also been shown to be associated with super-enhancers which can lead to overexpression of oncogenic driver genes such as BCL2, GFI1, and MYC. The development of a selective inhibitor of CDK7 is a promising strategy to control expression of these oncogenic genes in patients’ tumors. Previously, we described the design and synthesis of a potent and selective small molecule inhibitor of CDK7, TGN-1062, which was designed to bind to the catalytic ATP pocket. We showed efficacy in both AML and ovarian in vitro and in vivo models with correlative biomarker data. Here, we have generated further data on the effect and safety of TGN-1062 in pancreatic cancer cell line (CDX) and patient derived xenograft (PDX) models, both as a single agent and in combination with standard of care (SOC) therapies. Methods: In vivo efficacy studies were conducted in mice engrafted with pancreatic CDX and PDX tumors and treated with TGN-1062 with and without SOC therapies. Tumors were measured to determine tumor growth inhibition (TGI) and correlating drug accumulation levels were measured in both tumors and plasma using mass spectrometry. Immunohistochemistry (IHC) was performed on tumors to determine changes in cleaved Caspase-3, MYC, Ki67, and RNA Pol II. Rats were dosed with TGN-1062 to assess plasma exposures and preliminary tolerability. Results: In a pancreatic CDX model (MIA PaCa-2), TGN-1062 showed 38% TGI as a single agent and 121% TGI when in combination with gemcitabine and nab-paclitaxel, which on its own showed 37% TGI. In a pancreatic PDX model, TGN-1062 did not exhibit single agent effect, but combination with gemcitabine and nab-paclitaxel showed 95% TGI compared to 79% TGI with gemcitabine/nab-paclitaxel alone. Drug accumulation, biomarker analysis, and IHC from CDX and PDX tumors, showed that TGN-1062 treatment decreased RNA Pol II phosphorylation and MYC expression, and induced apoptosis. Rat pharmacokinetics indicated 100% oral bioavailability. Conclusion: Our potent and selective small molecule inhibitor of CDK7, TGN-1062, shows promise in pancreatic cancer models as a single or in combination with SOC. Preliminary data suggests that TGN-1062 can be developed further in multiple cancer indications. Citation Format: Alexis Weston, Trason Thode, Ryan Rodriguez del Villar, Serina Ng, Samuel Sampson, Shelby Rheinschmidt, Tithi Ghosh Halder, Raffaella Soldi, Mohan Kaadige, Srinivas Kasibhatla, Haiyong Han, Justin Moser, Vincent Chung, Joseph Chao, Victoria Villaflor, Daniel Von Hoff, Sunil Sharma. TGN-1062 inhibits CDK7 and augments the effects of gemcitabine and nab-paclitaxel in pancreatic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3931.

Evaluation of the Small-molecule BRD4 Degrader CFT-2718 in Small-cell Lung Cancer and Pancreatic Cancer Models.

Targeted, catalytic degradation of oncoproteins using heterobifunctional small molecules is an attractive modality, particularly for hematologic malignancies, which are often initiated by aberrant transcription factors and are challenging to drug with inhibitors. BRD4, a member of the bromodomain and extraterminal family, is a core transcriptional and epigenetic regulator that recruits the P-TEFb complex, which includes Cdk9 and cyclin T, to RNA polymerase II (pol II). Together, BRD4 and CDK9 phosphorylate serine 2 (pSer2) of heptad repeats in the C-terminal domain of RPB1, the large subunit of pol II, promote transcriptional elongation. Small-molecule degraders of BRD4 have shown encouraging efficacy in preclinical models for several tumor types but less efficacy in other cancers including small-cell lung cancer (SCLC) and pancreatic cancer. Here, we evaluated CFT-2718, a new BRD4-targeting degrader with enhanced catalytic activity and in vivo properties. In vivo, CFT-2718 has significantly greater efficacy than the CDK9 inhibitor dinaciclib in reducing growth of the LX-36 SCLC patient-derived xenograft (PDX) model and performed comparably to dinaciclib in limiting growth of the PNX-001 pancreatic PDX model. In vitro, CFT-2718 reduced cell viability in four SCLC and two pancreatic cancer models. In SCLC models, this activity significantly exceeded that of dinaciclib; furthermore, CFT-2718 selectively increased the expression of cleaved PARP, an indicator of apoptosis. CFT-2718 caused rapid BRD4 degradation and reduced levels of total and pSer2 RPB1 protein. These and other findings suggest that BRD-mediated transcriptional suppression merits further exploration in the setting of SCLC.

Oncological and genetic factors impacting PDX model construction with NSG mice in pancreatic cancer.

A patient-derived xenograft (PDX) approach, which relies on direct transplantation of tumor specimens into an immunocompromised animal, is a commonly used method for investigating tumor therapy predictions in vivo. This study evaluated influencing factors, including clinical, oncological, and genetic variables, for a pancreatic PDX model in mice. Tumor specimens were obtained from 121 patients with pancreatic ductal adenocarcinoma who underwent surgical resection at the Changhai Pancreatic Surgery Medical Center (Shanghai, China) between April 2016 and February 2017. Pancreatic cancer (PC) samples <3 mm3 were subcutaneously implanted into the NOD/Shi-scid/IL-2Rγnull (NSG) mice. Once the xenograft reached 300-500 mm3 or reached 180 d after cell inoculation, the tumor was excised. Part of the tumor was subsequently transplanted to next-generation mice, and another part was analyzed by using immunohistochemistry. Among the 121 patients with PC, tumor xenograft was successfully generated in 86 patients (71.1%). Primary tumor >3.5 cm in size was independently associated with xenograft formation rate. In addition, several enriched mutated genes within the VEGF pathway and higher microvessel density were found in the positive group (with xenograft) compared with the negative group (without xenograft). We concluded that tumor size and mutated VEGF pathway in PC are important factors affecting PDX model construction with NSG mice.-Guo, S., Gao, S., Liu, R., Shen, J., Shi, X., Bai, S., Wang, H., Zheng, K., Shao, Z., Liang, C., Peng, S., Jin, G. Oncological and genetic factors impacting PDX model construction with NSG mice in pancreatic cancer.

Abstract B050: Evaluation of TAK-264, a novel antibody-drug conjugate in pancreatic cancer cell lines and patient-derived xenograft models

Abstract Background: Antibody-drug conjugates (ADCs) are an emerging technology consisting of antibody, linker, and toxic agent, which have the potential to offer a more targeted therapeutic approach than standard chemotherapy regimens. ADCs selectively bind to antigens expressed in tumor environments. An emerging target for the treatment of pancreatic cancer is guanylyl cyclase C (GCC). GCC is a transmembrane G protein receptor found on the apical surface of intestinal enterocytes. It plays an important role in GI homeostasis. GCC is highly expressed in various colorectal carcinomas as well as adenocarcinomas of the upper GI tract. The objective of this study was to determine the antitumorigenic activity of TAK-264, an investigational ADC that targets GCC. Experimental Procedures: The antiproliferative effects of TAK-264 were assessed in a panel of eleven pancreatic cancer cell lines with various molecular backgrounds. Additionally, ten unique pancreatic ductal adenocarcinoma (PDAC) cancer patient-derived xenograft (PDX) models were treated with TAK-264 and the efficacy was determined. Tumor size was evaluated twice per week by caliper measurements. Sensitivity to TAK-264 was defined based on tumor growth inhibition that was statistically significant when compared to the vehicle control. Baseline levels of GCC were analyzed by IHC/immunoblotting and RT-PCR on PDX models and cell lines. Immunoblotting was performed to evaluate the effects of TAK-264 on downstream effectors. Results: GCC protein expression was analyzed by immunoblotting in normal versus tumor tissue; a marked increase in GCC expression was observed in tumor tissue when compared to matching normal tissue. The in vitro experiments demonstrated a range of responses to TAK-264 in an SRB assay. Eight of the ten PDAC PDX models demonstrated a statistically significant tumor growth inhibition when compared to the vehicle control. Immunoblotting demonstrated an increase in phosphorylated-Histone-H3 in two cell lines and in the PDAC PDX models treated with TAK-264, indicating a DNA damage response. The analysis of GCC protein expression in normal versus tumor tissue has shown a marked increase in GCC expression in tumor tissue when compared to matching normal tissue. There was no correlation between baseline levels of GCC and response to TAK-264 in either PDX or cell line models. Conclusions: TAK-264, an ADC targeting GCC, has good growth suppression activity in pancreatic cancer cell lines and in pancreatic PDX models. These findings support a hypothesis that further investigation of ADC targeting GCC may lead to novel therapeutic modalities for pancreatic cancer. Citation Format: Anna R. Schreiber, Anna Nguyen, Stacey M. Bagby, Betelehem Yacob, Kevin Quackenbush, Joe L. Guy, Thomas Crowell, Bradley Stringer, Hadi Danaee, Thea Kalebic, Wells A. Messersmith, John J. Arcaroli, Todd M. Pitts. Evaluation of TAK-264, a novel antibody-drug conjugate in pancreatic cancer cell lines and patient-derived xenograft models [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 B050.

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 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&amp;lt;0.001) and CEP1430 with gemcitabine selectively inhibited [80%] CSCs(n=50,p&amp;lt;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&amp;lt; 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 &amp;gt;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 &amp;gt; 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&amp;lt;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 &amp;gt;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

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.

Abstract 2639: Preclinical evaluation and biomarker identification for the anti-LGR5 mAb BNC101 in K-Ras mutant CRC and other solid tumor indications

Abstract BNC101 is a monoclonal antibody (mAb) targeting LGR5 that has successfully completed IND-enabling studies in preparation for Phase I clinical studies in 2015. LGR5 is a validated cancer stem cell (CSC) receptor overexpressed in colorectal cancer (CRC), pancreatic cancer and most other solid tumors. Loss and gain-of-function studies indicate that LGR5 is a functional cancer receptor involved in tumor growth and survival. Sorted LGR5+ primary CRC are highly tumorigenic compared to LGR5- cells in limiting dilution in vivo xenograft studies. BNC101 was selected as the lead LGR5 candidate for clinical development using Bionomics’ CSC Rx Discovery™ platform. Key lead selection criteria for BNC101 were anti-tumor and anti-CSC activity in vitro and in vivo, in patient-derived xenograft (PDX) tumor models from multiple tumor types. CSC activity was evaluated by functional in vivo limiting dilution assays (LDA) using tumor cells treated with BNC101 +/- standard-of-care chemotherapy (SOC) combinations. In LDA studies with the K-Ras mutant CRC PDX tumors CT1 and CT3, 5/8 (63%) and 6/8 (75%) mice implanted with serially diluted cells from BNC101 treated tumors remained tumor free, compared to 1/8 (13%) and 2/8 (25%) in the control groups. Combination with SOC further improved BNC101 activity against CT3 tumors, where 8/8 mice re-implanted for LDA had 6 months progression-free-survival (vs 3/8 in chemo group). In the pancreatic PDX model JH109, BNC101 combined with SOC flat-lined established tumors in 3/7 mice, compared to 0/7 tumors eradicated with control SOC alone. BNC101 is also active in multiple pancreatic, triple-negative breast (TNBC) and small-cell lung (SCLC) cancer xenograft models. Ongoing translational studies have identified a panel of Wnt genes modulated by BNC101 treatment that could potentially be used to identify BNC101 responsive versus resistant patients in the clinic. Furthermore, BNC101 treatment significantly reduced LGR5+ HLA+ circulating-tumor-cells (CTCs) in the peripheral blood of tumor-bearing mice, providing a pharmacodynamic (PD) biomarker of target engagement to further evaluate in clinical studies. Altogether, the preclinical data package supports our therapeutic hypothesis that BNC101 targeting of LGR5+ CSCs will significantly improve PFS and OS in solid tumor indications. Additional translational biomarker data to support the clinical development of BNC101 will also be presented. Citation Format: Peter Chu, Kristen Smith, Farbod Shojaei, Colin Walsh, John Norton, Jose Iglesias, Christopher Reyes. Preclinical evaluation and biomarker identification for the anti-LGR5 mAb BNC101 in K-Ras mutant CRC and other solid tumor indications. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2639. doi:10.1158/1538-7445.AM2015-2639

Transcriptional dissection of pancreatic tumors engrafted in mice.

BackgroundEngraftment of primary pancreas ductal adenocarcinomas (PDAC) in mice to generate patient-derived xenograft (PDX) models is a promising platform for biological and therapeutic studies in this disease. However, these models are still incompletely characterized. Here, we measured the impact of the murine tumor environment on the gene expression of the engrafted human tumoral cells.MethodsWe have analyzed gene expression profiles from 35 new PDX models and compared them with previously published microarray data of 18 PDX models, 53 primary tumors and 41 cell lines from PDAC. The results obtained in the PDAC system were further compared with public available microarray data from 42 PDX models, 108 primary tumors and 32 cell lines from hepatocellular carcinoma (HCC). We developed a robust analysis protocol to explore the gene expression space. In addition, we completed the analysis with a functional characterization of PDX models, including if changes were caused by murine environment or by serial passing.ResultsOur results showed that PDX models derived from PDAC, or HCC, were clearly different to the cell lines derived from the same cancer tissues. Indeed, PDAC- and HCC-derived cell lines are indistinguishable from each other based on their gene expression profiles. In contrast, the transcriptomes of PDAC and HCC PDX models can be separated into two different groups that share some partial similarity with their corresponding original primary tumors. Our results point to the lack of human stromal involvement in PDXs as a major factor contributing to their differences from the original primary tumors. The main functional differences between pancreatic PDX models and human PDAC are the lower expression of genes involved in pathways related to extracellular matrix and hemostasis and the up- regulation of cell cycle genes. Importantly, most of these differences are detected in the first passages after the tumor engraftment.ConclusionsOur results suggest that PDX models of PDAC and HCC retain, to some extent, a gene expression memory of the original primary tumors, while this pattern is not detected in conventional cancer cell lines. Expression changes in PDXs are mainly related to pathways reflecting the lack of human infiltrating cells and the adaptation to a new environment. We also provide evidence of the stability of gene expression patterns over subsequent passages, indicating early phases of the adaptation process.

Abstract B282: The PLK-4 inhibitor CFI-400945 reduces tumor growth in patient-derived pancreatic xenografts.

Abstract The polo-like kinase 4 (PLK-4) has recently emerged as a target that strongly correlates with aggressive tumor growth, high levels of hypoxia and metastasis in patient-derived pancreatic xenografts. In contrast to other members of the PLK family, the role of PLK-4 in cancer remains mostly elusive. Overexpression of PLK-4 in tumors has been suggested to contribute to chromosomal instability due to its crucial role in centriole duplication and therefore presents an interesting molecular target for the development of new therapeutic options for pancreatic cancer patients. The newly developed PLK-4 inhibitor CFI-400945 has been shown to specifically target PLK-4, resulting in incorrect centriole duplication and is currently developed for clinical trials. We used 6 orthotopically implanted patient-derived pancreatic xenograft models with varying growth rates and levels of tumor hypoxia in order to investigate the efficiency of the PLK-4 inhibitor CFI-400945 as a mono-therapy or in combination with Gemcitabine. Animals were treated with CFI-400945 using a daily dose of 7.5mg/kg for 21 days for experiments evaluating activity as a mono-therapy or with 13.5mg/kg according to a 2-day-on-5-days-off schedule in combination bi-weekly 100mg/kg Gemcitabine for 21 days. Tumor weight after completion of the treatment and overall survival were evaluated. Immunofluorescence (IF) staining of xenograft sections was used to evaluate PLK-4 inhibition, tumor hypoxia and proliferation. Treatment with CFI-400945 as a mono-therapy for 21 days significantly reduced tumor weight in 4 of the 6 tested patient-derived pancreatic xenograft models and an increase in overall survival in a number of responsive models. The strongest response to CFI-400945 treatment was observed in the highly hypoxic fast growing xenograft models OCIP51 where medium survival was 103days in the treatment group when compared to 54 days in vehicle treated animals. Experiments investigating the benefit of the combination of CFI-400945 with Gemcitabine using a clinically relevant treatment schedule are currently in progress in 2 xenograft models and will be completed shortly. We furthermore examined the correlation between tumor hypoxia, proliferation and response to CFI-400945 treatment in order to establish biological correlates for treatment response. Preliminary results show that although tumor weight was reduced in most of the tested models, the strongest response to CFI-400945 as a mono-therapy was observed in highly hypoxic models when compared to medium or low hypoxic models. These findings suggest that the PLK-4 inhibitor CFI-400945 may provide an effective treatment strategy for pancreatic cancer patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B282. Citation Format: Ines Lohse, Pinjiang Cao, Trevor Do, Emin Ibrahimov, Ming-Sound Tsao, David W. Hedley. The PLK-4 inhibitor CFI-400945 reduces tumor growth in patient-derived pancreatic xenografts. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B282.

Abstract 4504: PF-04449913, a small molecule inhibitor of Hedgehog signaling, is effective in inhibiting tumor growth in preclinical models

Abstract Aberrant activation of the Hedgehog (Hh) signaling pathway has been implicated in several human cancers. Mutations in the Patched (PTCH1) gene are responsible for basal cell nevus syndrome, and are commonly found in sporadic basal cell carcinoma and in medulloblastoma. In this study we evaluated PF-04449913, an inhibitor of the Hh signaling pathway, in a Ptch1+/-p53 mouse model of medulloblastoma and in human patient derived xenograft models. Treatment of Ptch1+/-p53+/- or Ptch1+/-p53-/- medulloblastoma allografts with PF-04449913 produced potent dose-dependent inhibition of Hh pathway activity resulting in stable tumor regression. Using Gli1 transcript levels as a surrogate for Hh pathway activity, the pharmacodynamic effects of PF-04449913 were evaluated in medulloblastoma allografts following single dose and multi dose administrations of compound. PF-04449913 treated medulloblastoma allografts had reduced levels of Gli1 gene expression and down regulation of genes linked to the Hh signaling pathway. PF-04449913 was also effective when combined with a chemotherapeutic agent in a colon patient derived xenograft model and a pancreatic patient derived xenograft model, resulting in 63% and 73% tumor growth inhibition respectively. Collectively, our study demonstrates the therapeutic efficacy of a small molecule inhibitor of Hh pathway in preclinical models of multiple cancer types in either single or combination treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4504. doi:10.1158/1538-7445.AM2011-4504

Abstract 4168: Comprehensive characterisation of a newly established patient-derived pancreatic adenocarcinoma xenograft collection

Abstract There is a high medical need to identify new treatments for patients with pancreatic cancer, since in many cases only palliative treatment is possible. The standard 1st line chemotherapy in inoperable, locally advanced (stage II and III) and metastatic (stage IV) adenocarcinoma of the pancreas is Gemcitabine as a single agent with a median survival of about 6 month. In addition, Gemcitabine is indicated as adjuvant chemotherapy after surgery. Although the anti-metabolite 5-Flourouracil (5-FU) and the EGF-R inhibitor Tarceva (Erlotinib) have been approved for 2nd line treatment, new and more efficient drugs are urgently needed. In the present study, more than 60 samples of pancreatic carcinomas were transplanted subcutaneously (s.c.) into NMRI nude mice directly after tumor resection. In most cases, tumor material from chemonaive patients with defined histology and staging was used for implantation. Up to now, 17 tumor models were passaged in nude mice and characterized comprehensively. In general, the histology of the primary tumor was comparable to that of the established xenograft. Chemosensitivity in vivo was evaluated by treatment of tumor bearing nude mice with 5-FU (100 or 75mg/kg, q7dx3, i.p.), Gemcitabine (240mg/kg, q7dx3, i.v.) and Erlotinib (25 and 50mg/kg, qdx21, p.o.). In general, tumor growth was not inhibited with best T/C values &amp;gt;50% highlighting the general chemoresistance of pancreatic cancer. Only for two models (PAXF 1872 and PAXF 1998), a high sensitivity towards Gemcitabine was evident with best T/C values of 8% and 3.8%, respectively. There was no correlation to the transcriptional levels of proteins involved in transportation and metabolism of Gemcitabine. Concerning Erlotinib, best T/C values were ranging from 89.6% (PAXF 1876) to 38.5% (PAXF 1982) with no correlation to EGFR expression status. A more broad chemosensitivity profile was established with the ex-vivo clonogenic assay. Interestingly, several tumors responded strongly to treatment with Rapamycin (IC50 ≤ 10nM). Based on this data, in vivo treatment experiments with RAD001 (Everolimus) were done and data are presented. Mutational analysis of p53 (exons 4 to 10) revealed only 4 out of 13 models as p53 wild-type. Nevertheless, the p53 pathway is dysregulated in all tumor models and for selected tumors, an aneuploid cell population was identified by ex vivo cell cycle analysis. In summary, a unique collection of patient-derived pancreatic xenograft models of high clinical relevance has been established. These models are available for translational research studies including in-vivo efficacy testing of new investigational drugs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4168.