PDF file - 242K, EGFR-overexpressing tumors respond after 1 week of treatment by H&E staining and IHC for total EGFR, phospho-EGFR, and Ki-67

PDF file - 325K, Nuclear p53 expression correlates positively with loss of wild type allele in serous epithelial ovarian carcinomas in p53m/+ mutant mice

PDF file - 117K, Reduction of p-EGFR and p-S6 in BIBW 2992 and rapamycin-treated lungs at 4, 8, and 24 hours post-treatment

PDF file - 117K, Reduction of p-EGFR and p-S6 in BIBW 2992 and rapamycin-treated lungs at 4, 8, and 24 hours post-treatment

PDF file - 92K, LC-MS/MS validation for selected metabolites

PDF file - 157K, Integrative data analysis of simultaneous measurement of metabolite and transcript concentrations in normal lung, lung tumors from untreated EGFR-TL mice, and lung tumors from vehicle, BIBW 2992, rapamycin, or BIBW 2992 plus rapamycin-treated EGFR-TL mice

PDF file - 267K, Widespread metabolomic alterations consistent with tumor development are found in blood of SEOC mice compared to wild type mice

PDF file - 161K, Analysis of recombination efficiency of targeted alleles in mutant mice

PDF file - 307K, lntrabursal injection of Adeno-Cre activates conditional alleles in OSE cells and induces histopathological changes of murine OSE post induction

PDF file - 253K, Gene expression analysis

PDF file - 53K, Tumor spectrum in Adeno-Cre injected mice

PDF file - 255K, Immunohistochemical staining for CA125, WT1 and calretinin

The desmoplastic stroma of pancreatic cancers forms a physical barrier that impedes intratumoral drug delivery. Attempts to modulate the desmoplastic stroma to increase delivery of administered chemotherapy have not shown positive clinical results thus far, and preclinical reports in which chemotherapeutic drugs were coadministered with antistromal therapies did not universally demonstrate increased genotoxicity despite increased intratumoral drug levels. In this study, we tested whether TGF beta antagonism can break the stromal barrier, enhance perfusion and tumoral drug delivery, and interrogated cellular and molecular mechanisms by which the tumor prevents synergism with coadministered gemcitabine. TGF beta inhibition in genetically engineered murine models (GEMM) of pancreas cancer enhanced tumoral perfusion and increased intratumoral gemcitabine levels. However, tumors rapidly adapted to TGF beta-dependent stromal modulation, and intratumoral perfusion returned to pre-treatment levels upon extended TGF beta inhibition. Perfusion was governed by the phenotypic identity and distribution of cancer-associated fibroblasts (CAF) with the myelofibroblastic phenotype (myCAFs), and myCAFs which harbored unique genomic signatures rapidly escaped the restricting effects of TGF beta inhibition. Despite the reformation of the stromal barrier and reversal of initially increased intratumoral exposure levels, TGF beta inhibition in cooperation with gemcitabine effectively suppressed tumor growth via cooperative reprogramming of T regulatory cells and stimulation of CD8 T cell-mediated antitumor activity. The antitumor activity was further improved by the addition of anti-PD-L1 immune checkpoint blockade to offset adaptive PD-L1 upregulation induced by TGF beta inhibition. These findings support the development of combined antistroma anticancer therapies capable of impacting the tumor beyond the disruption of the desmoplastic stroma as a physical barrier to improve drug delivery.

Synthetic host defense peptides (HDP) are a new class of promising therapeutic agents with potential application in a variety of diseases. RP-182 is a lOmer synthetic HDP design, which selectively reduces M2-like tumor associated macrophages via engagement with the cell surface lectin receptor MRC1/CD206 and is currently being developed as an innate immune defense regulator to improve anti-tumor immunity in immunologically cold tumors. Herein, we describe a sensitive and specific liquid chromatography (LC) coupled to quadrupole electron spray tandem mass spectrometry method to measure positively charged HDPs and HDP peptide fragments in complex biological matrices. Carboxylic acid magnetic beads were used as an affinity-capturing agent to extract the positively charged RP-182 from both mouse plasma and tissue homogenates. Beads were eluted with 0.1% (v/v) formic acid and chromatographic separation was achieved on a Waters 2.1 x 100 mm, 3.5 mu m XSelect Peptide CSH C18 column with a Vanguard precolumn of the same phase. MS/MS was performed on a Thermo TSQ Quantiva triple quadrupole mass spectrometer operating in Selected Reaction Monitoring (SRM) mode fragmenting the plus three parent ion 458.9(+3) and monitoring ions 624.0(+2), 550.5(+2), and 597.3(+1) for RP-182 and 462.4(+3) > 629.1(+2), 555.5(+2), and 607.3(+1) for isotopic RP-182 standard. The assay had good linearity ranging from 1 ng to 1000 ng in mouse plasma with the lower limit of detection for RP-182 at 1 ng in mouse plasma with good intra- and inter-sample precision and accuracy. Recovery ranged from 66% to 77% with minimum matrix effects. The method was successfully applied to an abbreviated pharmacokinetic study in mice after single IP injection of RP-182. The method was successfully tested on a second HDP, the 17mer D4E1, and the cationic human peptide hormone ghrelin suggesting that it might be a general sensitive method applicable to quantifying HDP peptides that are difficult to extract. Published by Elsevier B.V.

Metarrestin is a first-in-class small molecule clinical candidate capable of disrupting the perinucleolar compartment, a subnuclear structure unique to metastatic cancer cells. This study aims to define the pharmacokinetic (PK) profile of metarrestin and the pharmacokinetic/pharmacodynamic relationship of metarrestin-regulated markers.

The high mortality rate from ovarian cancers can be attributed to late-stage diagnosis and lack of effective treatment. Despite enormous effort to develop better targeted therapies, platinum-based chemotherapy still remains the standard of care for ovarian cancer patients, and resistance occurs at a high rate. One of the rate limiting factors for translation of new drug discoveries into clinical treatments has been the lack of suitable preclinical cancer models with high predictive value. We previously generated genetically engineered mouse (GEM) models based on perturbation of Tp53 and Rb with or without Brca1 or Brca2 that develop serous epithelial ovarian cancer (SEOC) closely resembling the human disease on histologic and molecular levels. Here, we describe an adaptation of these GEM models to orthotopic allografts that uniformly develop tumors with short latency and are ideally suited for routine preclinical studies. Ovarian tumors deficient in Brca1 respond to treatment with cisplatin and olaparib, a PARP inhibitor, whereas Brca1-wild type tumors are non-responsive to treatment, recapitulating the relative sensitivities observed in patients. These mouse models provide the opportunity for evaluation of effective therapeutics, including prediction of differential responses in Brca1-wild type and Brca1-deficient tumors and development of relevant biomarkers.

Abstract Patients with lung cancer with activating mutations in the EGF receptor (EGFR) kinase, who are treated long-term with tyrosine kinase inhibitors (TKI), often develop secondary mutations in EGFR associated with resistance. Mice engineered to develop lung adenocarcinomas driven by the human EGFR T790M resistance mutation are similarly resistant to the EGFR TKI erlotinib. By tumor volume endpoint analysis, these mouse tumors respond to BIBW 2992 (an irreversible EGFR/HER2 TKI) and rapamycin combination therapy. To correlate EGFR-driven changes in the lung with response to drug treatment, we conducted an integrative analysis of global transcriptome and metabolite profiling compared with quantitative imaging and histopathology at several time points during tumor progression and treatment. Responses to single-drug treatments were temporary, whereas combination therapy elicited a sustained response. During tumor development, metabolomic signatures indicated a shift to high anabolic activity and suppression of antitumor programs with 11 metabolites consistently present in both lung tissue and blood. Combination drug treatment reversed many of the molecular changes found in tumored lung. Data integration linking cancer signaling networks with metabolic activity identified key pathways such as glutamine and glutathione metabolism that signified response to single or dual treatments. Results from combination drug treatment suggest that metabolic transcriptional control through C-MYC and SREBP, as well as ELK1, NRF1, and NRF2, depends on both EGFR and mTORC1 signaling. Our findings establish the importance of kinetic therapeutic studies in preclinical assessment and provide in vivo evidence that TKI-mediated antiproliferative effects also manifest in specific metabolic regulation. Cancer Res; 72(22); 5921–33. ©2012 AACR.

The majority of human high-grade serous epithelial ovarian cancer (SEOC) is characterized by frequent mutations in p53 and alterations in the RB and FOXM1pathways. A subset of humanSEOCharbors a combination of germline and somatic mutations as well as epigenetic dysfunction for BRCA1/2. Using Cre-conditional alleles and intrabursal induction by Cre-expressing adenovirus in genetically engineered mice, we analyzed the roles of pathway perturbations in epithelial ovarian cancer initiation and progression. Inactivation of RB-mediated tumor suppression induced surface epithelial proliferation with progression to stage I carcinoma. Additional biallelic inactivation and/or missense p53 mutation in the presence or absence of Brca1/2 caused progression to stage IV disease. As in human SEOC,mice developed peritoneal carcinomatosis, ascites, and distantmetastases. Unbiased gene expression and metabolomic profiling confirmed that Rb, p53, and Brca1/2-triple mutant tumors aligned with human SEOC, and notwith other intraperitoneal cancers. Together, ourfindings provide a novel resource for evaluating disease etiology and biomarkers, therapeutic evaluation, and improved imaging strategies in epithelial ovarian cancer. Cancer Res; 72(16); 4141–53. 2012 AACR.

Abstract The majority of human high-grade serous epithelial ovarian cancer (SEOC) is characterized by frequent mutations in p53 and alterations in the RB and FOXM1 pathways. A subset of human SEOC harbors a combination of germline and somatic mutations as well as epigenetic dysfunction for BRCA1/2. Using Cre-conditional alleles and intrabursal induction by Cre-expressing adenovirus in genetically engineered mice, we analyzed the roles of pathway perturbations in epithelial ovarian cancer initiation and progression. Inactivation of RB-mediated tumor suppression induced surface epithelial proliferation with progression to stage I carcinoma. Additional biallelic inactivation and/or missense p53 mutation in the presence or absence of Brca1/2 caused progression to stage IV disease. As in human SEOC, mice developed peritoneal carcinomatosis, ascites, and distant metastases. Unbiased gene expression and metabolomic profiling confirmed that Rb, p53, and Brca1/2-triple mutant tumors aligned with human SEOC, and not with other intraperitoneal cancers. Together, our findings provide a novel resource for evaluating disease etiology and biomarkers, therapeutic evaluation, and improved imaging strategies in epithelial ovarian cancer. Cancer Res; 72(16); 4141–53. ©2012 AACR.