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PDF file - 239KB, Western blot showed inhibition of autophagy in Atg5 and Atg7 knockdown cell lines.

PDF file, 209K, Representative H&E stained sections from Panc374 xenograft.

XLSX file, 105K, Gene expression analysis for predictive signatures of response to MRK-003.

Supplementary Figure 2 from Impact of APE1/Ref-1 Redox Inhibition on Pancreatic Tumor Growth

PDF file - 162KB, PCR and western blot confirmed the genotype of mouse cell lines with different p53 status.

Supplementary Table 2 from A Pilot Clinical Study of Treatment Guided by Personalized Tumorgrafts in Patients with Advanced Cancer

Supplementary Figure 1 from A Pilot Clinical Study of Treatment Guided by Personalized Tumorgrafts in Patients with Advanced Cancer

Supplementary Methods, Figure Legends 1-3 from Impact of APE1/Ref-1 Redox Inhibition on Pancreatic Tumor Growth

Patients with many advanced solid cancers have very poor prognosis, and improvements in life expectancy are measured only in months. We have recently reported the remarkable clinical outcome of a patient with advanced, gemcitabine-resistant, pancreatic cancer who was later treated with DNA-damaging agents, on the basis of the observation of significant activity of this class of drugs against a personalized tumorgraft generated from the patient's surgically resected tumor. Here, we extend the approach to patients with other advanced cancers. Tumors resected from 14 patients with refractory advanced cancers were propagated in immunodeficient mice and treated with 63 drugs in 232 treatment regimens. An effective treatment regimen in the xenograft model was identified for 12 patients. One patient died before receiving treatment, and the remaining 11 patients received 17 prospectively guided treatments. Fifteen of these treatments resulted in durable partial remissions. In 2 subjects, no effective treatments were found. Overall, there was a remarkable correlation between drug activity in the model and clinical outcome, both in terms of resistance and sensitivity. The data support the use of the personalized tumorgraft model as a powerful investigational platform for therapeutic decision making and to efficiently guide cancer treatment in the clinic. Mol Cancer Ther; 10(8); 1311–6. ©2011 AACR.

PDF file - 181KB, Deletion of Atg5 in pancreata disrupts beta-islets over time.

Pancreatic cancer is especially a deadly form of cancer with a survival rate less than 2%. Pancreatic cancers respond poorly to existing chemotherapeutic agents and radiation, and progress for the treatment of pancreatic cancer remains elusive. To address this unmet medical need, a better understanding of critical pathways and molecular mechanisms involved in pancreatic tumor development, progression, and resistance to traditional therapy is therefore critical. Reduction–oxidation (redox) signaling systems are emerging as important targets in pancreatic cancer. AP endonuclease1/Redox effector factor 1 (APE1/Ref-1) is upregulated in human pancreatic cancer cells and modulation of its redox activity blocks the proliferation and migration of pancreatic cancer cells and pancreatic cancer-associated endothelial cells in vitro. Modulation of APE1/Ref-1 using a specific inhibitor of APE1/Ref-1′s redox function, E3330, leads to a decrease in transcription factor activity for NFκB, AP-1, and HIF1α in vitro. This study aims to further establish the redox signaling protein APE1/Ref-1 as a molecular target in pancreatic cancer. Here, we show that inhibition of APE1/Ref-1 via E3330 results in tumor growth inhibition in cell lines and pancreatic cancer xenograft models in mice. Pharmacokinetic studies also show that E3330 attains more than10 μmol/L blood concentrations and is detectable in tumor xenografts. Through inhibition of APE1/Ref-1, the activity of NFκB, AP-1, and HIF1α that are key transcriptional regulators involved in survival, invasion, and metastasis is blocked. These data indicate that E3330, inhibitor of APE1/Ref-1, has potential in pancreatic cancer and clinical investigation of APE1/Ref-1 molecular target is warranted. Mol Cancer Ther; 10(9); 1698–708. ©2011 AACR.

PDF file, 76K, FACS plots for ALDH and CD44 and CD24 controls.

XLSX file, 138K, Gene expression analysis for predictive signatures of response to MRK-003 plus GEM.

PDF file - 283KB, Western blot on tumor lines derived from primary tumors confirmed Atg5 status and also showed loss of p53.

PDF file - 18KB, Summary of genetic mutations in different human pancreatic tumor xenografts.

PDF file - 337KB, IHC staining shows absence of Atg5 expression and diminished autophagosomes in tumors with Atg5 deletion.

Supplementary Figure 1 from Impact of APE1/Ref-1 Redox Inhibition on Pancreatic Tumor Growth

PDF file, 186K, Baseline gene expression profiles identify signatures of response to single agent MRK-003 and the combination of MRK-003 and GEM.

Supplementary Table 1 from A Pilot Clinical Study of Treatment Guided by Personalized Tumorgrafts in Patients with Advanced Cancer

Purpose: To determine the efficacy of AZD0530, an orally active small molecule Src inhibitor, in human pancreatic cancer xenografts and to seek biomarkers predictive of activity.Experimental Design: Sixteen patient-derived pancreatic cancer xenografts from the PancXenoBank collection at Johns Hopkins were treated with AZD0530 (50 mg/kg/day, p.o.) for 28 days. Baseline gene expression profiles of differently expressed genes in 16 tumors by Affymetrix U133 Plus 2.0 gene array were used to predict AZD0530 sensitivity in an independent group of eight tumors using the K-Top Scoring Pairs (K-TSP) method.Results: Three patient tumors of 16 were found to be sensitive to AZD0530, defined as tumor growth K-TSP classifier identified one gene pair (LRRC19 and IGFBP2) from the 16 training cases based on a decision rule. The classifier achieved 100% and 83.3% of sensitivity and specificity in an independent test set that consists of eight xenograft cases.Conclusions: AZD0530 treatment significantly inhibits the tumor growth in a subset of human pancreatic tumor xenografts. One gene pair (LRRC19 and IGFBP2) identified by the K-TSP classifier has high predictive power for AZD0530 sensitivity, suggesting the potential for this gene pair as biomarker for pancreatic tumor sensitivity to AZD0530.

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Purpose: The goal of this study was to evaluate prospectively the engraftment rate, factors influencing engraftment, and predictability of clinical outcome of low-passage xenografts from patients with resectable pancreatic ductal adenocarcinoma (PDA) and to establish a bank of PDA xenografts.Experimental Design: Patients with resectable PDA scheduled for resection at the Johns Hopkins Hospital were eligible. Representative pieces of tumor were implanted in nude mice. The status of the SMAD4 gene and content of tumor-generating cells were determined by immunohistochemistry. Gene expression was carried out by using a U133 Plus 2.0 array. Patients were followed for progression and survival.Results: A total of 94 patients with PDA were resected, 69 tumors implanted in nude mice, and 42 (61%) engrafted. Engrafted carcinomas were more often SMAD4 mutant, and had a metastatic gene expression signature and worse prognosis. Tumors from patients resistant to gemcitabine were enriched in stroma-related gene pathways. Tumors sensitive to gemcitabine were enriched in cell cycle and pyrimidine gene pathways. The time to progression for patients who received treatment with gemcitabine for metastatic disease (n = 7) was double in patients with xenografts sensitive to gemcitabine.Conclusion: A successful xenograft was generated in 61% of patients attempted, generating a pool of 42 PDA xenografts with significant biological information and annotated clinical data. Patients with PDA and SMAD4 inactivation have a better engraftment rate. Engraftment is a poor prognosis factor, and engrafted tumors have a metastatic gene expression signature. Tumors from gemcitabine-resistant patients were enriched in stromal pathways. Clin Cancer Res; 17(17); 5793–800. ©2011 AACR.

Supplementary Figures 1-7 and Supplementary Table 1. Supplementary Figure 1 H&E stain and trichrome stain of representative 7-week-old KIC tumor tissue. Supplementary Figure 2 Axl gene expression in pancreatic cancer patient-derived xenografts. Supplementary Figure 3 Immunofluorescence of cleaved-caspase 3, p-histon-3, endomucin and EMT markers in Pan02 tumor tissue. Supplementary Figure 4 Western blot of TBK1/NF-κB signaling in tumor lysates from KIC mice treated with/without BGB324. Supplementary Figure 5 F4/80 stain in tumor tissue from KIC mice treated with BGB324 +/- gemcitabine. Supplementary Figure 6 Arginase 1 mRNA expression level in bone marrow-derived macrophages treated with IL-4 +/- BGB324. Supplementary Figure 7 Flow cytometry of KPC-M09 subcutaneous tumors treated with/without BGB324. Supplementary Table 1 Cytokine/Chemokine Concentration in KIC tumor lysates from different treatment groups.

SF 1. Heat map showing the tumor growth inhibition (TGI) of six metabolic inhibitors in PDX panel. SF 2. Tumor growth curves of PDXs treated with phenformin. SF 3. Tumor doubling time of 12 individual PDXs. SF 4. Aggregate tumor doubling time of PDXs treated with vehicle or phenformin.

PDF file - 70K, We examined a gene signature of response to gemcitabine that included genes involved in the uptake (nucleoside transporters) and activation of gemcitabine (deoxycytin kinase) in tumor samples from patient JH034. We included samples from primary tumor, subcutaneous and orthotopic xenografts. Our analysis shows that primary and orthotopic xenografts are clustered together and differ from subcutaneous xenografts.

Supplementary Fig. S3 from Efficacy and pharmacodynamic effects of bosutinib (SKI-606), a Src/Abl inhibitor, in freshly generated human pancreas cancer xenografts

Expanded Methods

Supplementary Data from Antitumor Effects and Biomarkers of Activity of AZD0530, a Src Inhibitor, in Pancreatic Cancer

PDF file - 115K

Recently, Src tyrosine kinase has emerged as an attractive target for anticancer therapy, and Src is overexpressed in pancreatic cancer. The purpose of the study was to investigate the in vivo efficacy and pharmacodynamic effects of bosutinib (SKI-606), a Src/Abl inhibitor, using a panel of human pancreatic tumor xenografts. Surgically resected human pancreatic tumors were implanted into female nude mice and randomized to bosutinib versus control. Src and other pathways were analyzed by Western Blot, IHC, and Affymetrix U133 Plus 2.0 gene arrays. Of 15 patient tumors, 3 patient tumors were found to be sensitive to bosutinib, defined as tumor growth of

Purpose: To identify effective metabolic inhibitors to suppress the aggressive growth of pancreatic ductal adenocarcinoma (PDAC), we explored the in vivo antitumor efficacy of metabolic inhibitors, as single agents, in a panel of patient-derived PDAC xenograft models (PDX) and investigated whether genomic alterations of tumors correlate with the sensitivity to metabolic inhibitors.Experimental Design: Mice with established PDAC tumors from 6 to 13 individual PDXs were randomized and treated, once daily for 4 weeks, with either sterile PBS (vehicle) or the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), transaminase inhibitor aminooxyacetate (AOA), pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA), autophagy inhibitor chloroquine (CQ), and mitochondrial complex I inhibitor phenformin/metformin.Results: Among the agents tested, phenformin showed significant tumor growth inhibition (>30% compared with vehicle) in 5 of 12 individual PDXs. Metformin, at a fivefold higher dose, displayed significant tumor growth inhibition in 3 of 12 PDXs similar to BPTES (2/8 PDXs) and DCA (2/6 PDXs). AOA and CQ had the lowest response rates. Gene set enrichment analysis conducted using the baseline gene expression profile of pancreatic tumors identified a gene expression signature that inversely correlated with phenformin sensitivity, which is in agreement with the phenformin gene expression signature of NIH Library of Integrated Network-based Cellular Signatures (LINCS). The PDXs that were more sensitive to phenformin showed a baseline reduction in amino acids and elevation in oxidized glutathione. There was no correlation between phenformin response and genetic alterations in KRAS, TP53, SMAD4, or PTEN.Conclusions: Phenformin treatment showed relatively higher antitumor efficacy against established PDAC tumors, compared with the efficacy of other metabolic inhibitors and metformin. Phenformin treatment significantly diminished PDAC tumor progression and prolonged tumor doubling time. Overall, our results serve as a foundation for further evaluation of phenformin as a therapeutic agent in pancreatic cancer. Clin Cancer Res; 23(18); 5639–47. ©2017 AACR.

Supplementary Figure 1 from Coordinated Epidermal Growth Factor Receptor Pathway Gene Overexpression Predicts Epidermal Growth Factor Receptor Inhibitor Sensitivity in Pancreatic Cancer

Supplementary Figures and Table legends

Supplementary Table 1 from Coordinated Epidermal Growth Factor Receptor Pathway Gene Overexpression Predicts Epidermal Growth Factor Receptor Inhibitor Sensitivity in Pancreatic Cancer

Supplementary Table 2 from Coordinated Epidermal Growth Factor Receptor Pathway Gene Overexpression Predicts Epidermal Growth Factor Receptor Inhibitor Sensitivity in Pancreatic Cancer

Supplementary Figure 1. Baseline TIGAR mRNA expression in tumors.

The epidermal growth factor receptor (EGFR) inhibitor erlotinib is approved for treatment of pancreatic cancer but the overall activity is minimal, and known predictive factors for EGFR inhibitor efficacy are infrequent in this disease. We tested the hypothesis that global activation of the EGFR pathway is predictive of EGFR inhibitor efficacy. Pancreatic cancer tumors directly xenografted at surgery were treated with the EGFR inhibitors erlotinib and cetuximab and analyzed for biological features. Two of 10 tumors were sensitive, and by global gene expression profiling with gene set enrichment analysis, the EGFR pathway was highly expressed in sensitive compared with resistant tumors. The core gene components driving EGFR pathway overexpression were pathway ligands and positive effectors. In a prospective validation, the EGFR pathway-based signature correctly predicted anti-EGFR treatment response in eight additional tumors and was not predictive of response to gemcitabine and CI1040 (a MEK inhibitor). Analysis of EGFR, KRAS, and PIK3CA mutations and gene amplification by fluorescence in situ hybridization and multiplex ligation-dependent probe amplification showed that none of these genetic abnormalities were neither predictive nor responsible for the EGFR pathway activation. Coordinated overexpression of the EGFR pathway predicts susceptibility to EGFR inhibitors in pancreatic cancer. These results suggest a phenomenon of pathway addiction and support the value of unbiased system biology approaches in drug development. [Cancer Res 2008;68(8):2841–9]

Polymorphism drives survival under stress and provides adaptability. Genetic polymorphism of ribosomal RNA (rRNA) genes derives from internal repeat variation of this multicopy gene, and from interindividual variation. A considerable amount of rRNA sequence heterogeneity has been proposed but has been challenging to estimate given the scarcity of accurate reference sequences. We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling and used high-coverage whole-genome sequencing (WGS) data from the 1000 Genomes Project to analyze variants in 2504 individuals from 26 populations. We identified a total of 3791 variant positions. The variants positioned nonrandomly on the rRNA gene. Invariant regions included the promoter, early 5′ ETS, most of 18S, 5.8S, ITS1, and large areas of the intragenic spacer. A total of 470 variant positions were observed on 28S rRNA. The majority of the 28S rRNA variants were located on highly flexible human-expanded rRNA helical folds ES7L and ES27L, suggesting that these represent positions of diversity and are potentially under continuous evolution. Several variants were validated based on RNA-seq analyses. Population analyses showed remarkable ancestry-linked genetic variance and the presence of both high penetrance and frequent variants in the 5′ ETS, ITS2, and 28S regions segregating according to the continental populations. These findings provide a genetic view of rRNA gene array heterogeneity and raise the need to functionally assess how the 28S rRNA variants affect ribosome functions.