Faculty Bibliography

Background: Pancreatic cancer is well known for its aggressive clinical course and resistance to chemotherapy. A Phase I trial of CD40 immunotherapy in combination with gemcitabine demonstrated the combination was safe and achieves tumor responses in patients with pancreatic ductal adenocarcinoma. We investigated the effectiveness of gemcitabine, albumin-bound paclitaxel and CD40 agonist immunotherapy in an orthotopic pancreatic mouse model. Methods: Pancreatic cells obtained from a Kras^G12D;Trp53^R172H (KPC) genetically engineered mouse were cultivated in cell culture and surgically implanted into the pancreata of immunocompetent syngeneic C57/Bl6 mice allowing for tumor formation in situ. Two weeks after KPC cell implantation, mice were treated with 120 mg/kg gemcitabine and 120 mg/kg nab-paclitaxel by intraperitoneal injection. Forty eight hours after chemotherapy administration, mice were treated with 100 ug of FGK45 CD40 immunotherapy. Mouse tumors and spleens were harvested from euthanized mice ten days after drug treatment. Tumor and spleens were analyzed histologically and by flow cytometry. Results: Mice treated with combination chemotherapy and immunotherapy had a significant reduction in tumor volume in comparison to vehicle treated mice. Combination chemotherapy did not cause a significant decrease in tumor volume. No changes were seen in stromal remodeling using trichrome histological staining. Mice treated with CD40 immunotherapy had an increase in spleen size indicating an immune response. Histological and flow cytometry analysis revealed an increase in CD45+ cells in the tumors of the CD40 immunotherapy treated samples in comparison to chemotherapy alone. Conclusions: CD40 immunotherapy in combination with gemcitabine and albumin-bound paclitaxel has significant antitumor activity in an orthotopic pancreatic cancer mouse model provoking an immune response in the tumors. Future experiments will focus on identifying immune mediators critical for drug efficacy

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive tumor stroma that is composed of immune cells, vascular cells, fibroblasts and extracellular matrix components (Erkan et al., 2012). This increased desmoplasia correlates with decreased survival and has been shown to mediate chemoresistance (von Hoff et al., 2009). Macrophages within the stromal compartment have been shown to play tumor-promoting roles by switching from their classical immunostimulatory function ('M1') to an immunosuppressive state ('M2') (Yoshikawa et al., 2012). Nab-Paclitaxel (nab-Ptx) is an albumin-bound form of Paclitaxel that, in combination with Gemcitabine, is currently accepted as the standard chemotherapeutic regimen for pancreatic cancer. However, pre-clinical and clinical studies have suggested that nab-Ptx may exert its effects by altering the tumor stroma (Desai et al., 2006, von Hoff et al., 2011). Here, we show that tumor-associated macrophages uptake high levels of nab-Ptx in an orthotopic model of PDAC via macropinocytosis. Eighty to ninety percent of macrophages within the tumor internalize fluorescently labeled nab-Ptx ex vivo, as compared to thirty to forty percent of macrophages from the spleens of the same animals. These data suggest that M2-like macrophages within the tumor microenvironment preferentially macropinocytose nab-Ptx. To analyze the potential consequence of nab-Ptx internalization on macrophages, we treated the RAW macrophage cell line with Ptx alone or in combination with the immunostimulatory cytokine IFN gamma (IFNgamma). Prolonged exposure (48h) of RAW cells to Ptx induced an increase in the M1 marker iNOS, with the combination of low dose IFNgamma and Ptx resulting in a synergistic increase in iNOS expression with accelerated kinetics (12h). Moreover, Ptx alone or in combination with IFNgamma was able to revert IL-4-induced expression of the M2 marker Arginase 1. These findings suggest that high levels of nab-Ptx internalization by M2 macrophages may re-polarize them to an M1, immunostimulatory state. Our in vitro studies suggest that the potential effect of nab-Ptx and Gemcitabine on macrophage polarization in vivo may be enhanced by the presence of an additional immunostimulatory signal. The agonist CD40 monoclonal antibody (mAb) is a member on the TNFalpha receptor superfamily that has been shown to induce immune cell activation and therapeutic efficacy in human and mouse models of PDAC (Beatty et al., 2013). We therefore examined the effect of combining CD40 mAb with nab-Ptx and Gemcitabine treatment on macrophage phenotype in an orthotopic model of PDAC. Our studies to date show that the induction of both an increase in M1 marker (MHCII and CD86) expression and a decrease in M2 marker expression (CD206) in pancreas tumor-associated macrophages requires the triple combination therapy. Together, these data suggest that the internalization of nab-Ptx by tumor-associated macrophages in combination with immune activating signals like CD40 mAb may be required to restore their M1-like, tumor cell cytotoxic functions. CD40 mAb and nab-Ptx combination therapy may therefore enable the effective targeting of the pancreatic tumor stroma, resulting in enhanced therapeutic benefit in PDAC

Tumor graft models (also known as patient-derived xenografts or PDX) are based on the transfer of primary tumors directly from the patient into an immunodeficient mouse. Because PDX mice are derived from human tumors, they offer a tool for developing anticancer therapies and personalized medicine for patients with cancer. In addition, these models can be used to study metastasis and tumor genetic evolution. This review examines the development, challenges, and broad use of these attractive preclinical models.

RNAi screening holds the promise of systemizing the search for combination therapeutic strategies. Here we performed a pooled shRNA library screen to look for promising targets to inhibit in combination with inhibition of the mitotic regulator polo-like kinase (PLK1). The library contained ~4,500 shRNAs targeting various signaling and cancer-related genes and was screened in four lung cancer cell lines using both high (IC80) and low (IC20) amounts of the PLK1 inhibitor GSK461364. The relative abundance of cells containing individual shRNAs following drug treatment was determined by microarray analysis, using the mock treatment replicates as the normalizing reference. Overall, the inferred influences of individual shRNAs in both high and low drug treatment were remarkably similar in all four cell lines and involved a large percentage of the library. To investigate which functional categories of shRNAs were most prominent in influencing drug response, we used statistical analysis of microarrays (SAM) in combination with a filter for genes that had two or more concordant shRNAs. The most significant functional categories that came out of this analysis included receptor tyrosine kinases and nuclear hormone receptors. Through individual validation experiments, we determined that the two shRNAs from the library targeting the nuclear retinoic acid receptor gene RARA did indeed silence RARA expression and as predicted conferred resistance to GSK461364. This led us to test whether activation of RARA receptor with retinoids could sensitize cells to GSK461364. We found that retinoids did increase the drug sensitivity and enhanced the ability of PLK1 inhibition to induce mitotic arrest and apoptosis. These results suggest that retinoids could be used to enhance the effectiveness of GSK461364 and provide further evidence that RNAi screens can be effective tools to identify combination target strategies.

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

Designing potent silencing triggers is key to the successful application of RNA interference (RNAi) in mammals. Recent studies suggest that the assembly of RNAi effector complexes is coupled to Dicer cleavage. Here we examine whether transfection of optimized Dicer substrates results in an improved RNAi response. Dicer cleavage of chemically synthesized short hairpin RNAs (shRNAs) with 29-base-pair stems and 2-nucleotide 3' overhangs produced predictable homogeneous small RNAs comprising the 22 bases at the 3' end of the stem. Consequently, direct comparisons of synthetic small interfering RNAs and shRNAs that yield the same small RNA became possible. We found synthetic 29-mer shRNAs to be more potent inducers of RNAi than small interfering RNAs. Maximal inhibition of target genes was achieved at lower concentrations and silencing at 24 h was often greater. These studies provide the basis for an improved approach to triggering experimental silencing via the RNAi pathway.