Camilla L. Christensen

Activation of the PD-1 Pathway Contributes to Immune Escape in EGFR-Driven Lung Tumors

UNLABELLED The success in lung cancer therapy with programmed death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between EGF receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, CTL antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased CTLs and increased markers of T-cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T-cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape and mechanistically link treatment response to PD-1 inhibition. SIGNIFICANCE We show that autochthonous EGFR-driven lung tumors inhibit antitumor immunity by activating the PD-1/PD-L1 pathway to suppress T-cell function and increase levels of proinflammatory cytokines. These findings indicate that EGFR functions as an oncogene through non-cell-autonomous mechanisms and raise the possibility that other oncogenes may drive immune escape.

Viral vector-mediated gene expression in olfactory ensheathing glia implants in the lesioned rat spinal cord

Implantation of olfactory ensheathing glia (OEG) is a promising strategy to augment long-distance regeneration in the injured spinal cord. In this study, implantation of OEG following unilateral hemisection of the dorsal cervical spinal cord was combined with ex vivo gene transfer techniques. We report, to our knowledge for the first time, that purified cultures of primary OEG are capable of expressing a foreign gene following adenoviral (AdV) and lentiviral (LV) vector-mediated gene transfer. OEG implants subjected to AdV vector-mediated gene transfer expressed high levels of transgenic protein in both intact and lesioned spinal cord at 7 days after implantation. However, the levels of transgene expression gradually declined between 7 and 30 days after implantation in lesioned spinal cord. Infection with LV vectors resulted in stable transduction of primary OEG cultures and transgene expression persisted for at least 4 months after implantation. Genetic engineering of OEG opens the possibility of expressing additional neurotrophic genes and create optimal ‘bridging’ substrates to support spinal axon regeneration. Furthermore, stable transduction of OEG allows us to reliably study the behaviour of implanted cells and to obtain better understanding of their regeneration supporting properties.

In vitro and in vivo effects of polyethylene glycol (PEG)-modified lipid in DOTAP/cholesterol-mediated gene transfection

Background: DOTAP/cholesterol-based lipoplexes are successfully used for delivery of plasmid DNA in vivo especially to the lungs, although low systemic stability and circulation have been reported. To achieve the aim of discovering the best method for systemic delivery of DNA to disseminated tumors we evaluated the potential of formulating DOTAP/cholesterol lipoplexes with a polyethylene glycol (PEG)-modified lipid, giving the benefit of the shielding and stabilizing properties of PEG in the bloodstream. Method: A direct comparison of properties in vitro and in vivo of 4 different DOTAP/cholesterol-based lipoplexes containing 0%, 2%, 4%, and 10% PEG was performed using reporter gene activity and radioactive tracer lipid markers to monitor biodistribution. Results: We found that 10% PEGylation of lipoplexes caused reduced retention in lung and heart tissues of nude mice compared to nonPEGylated lipoplexes, however no significant delivery to xenograft flank tumors was observed. Although PEGylated and nonPEGylated lipoplexes were delivered to cells the ability to mediate successful transfection is hampered upon PEGylation, presumably due to a changed uptake mechanism and intracellular processing. Conclusion: The eminent in vivo transfection potency of DOTAP/cholesterol-based lipoplexes is well established for expression in lung tumors, but it is unsuitable for expression in non first pass organs such as xenograft flank tumors in mice even after addition of a PEG-lipid in the formulation.

PRIMA-1Met/APR-246 induces apoptosis and tumor growth delay in small cell lung cancer expressing mutant p53

Purpose: Small cell lung cancer (SCLC) is a highly malignant disease with poor prognosis, necessitating the need to develop new and efficient treatment modalities. PRIMA-1Met (p53-dependent reactivation of massive apoptosis), also known as APR-246, is a small molecule, which restores tumor suppressor function to mutant p53 and induces cancer cell death in various cancer types. Since p53 is mutated in more than 90% of SCLC, we investigated the ability of PRIMA-1Met to induce apoptosis and inhibit tumor growth in SCLC with different p53 mutations. Experimental Design: The therapeutic effect of PRIMA-1Met/APR-246 was studied in SCLC cells in vitro using cell viability assay, fluorescence-activated cell-sorting analysis, p53 knockdown studies, and Western blot analyses. The antitumor potential of PRIMA-1Met/APR-246 was further evaluated in two different SCLC xenograft models. Results: PRIMA-1Met/APR-246 efficiently inhibited the growth of the SCLC cell lines expressing mutant p53 in vitro and induced apoptosis, associated with increased fraction of cells with fragmented DNA, caspase-3 activation, PARP cleavage, Bax and Noxa upregulation and Bcl-2 downregulation in the cells. The growth suppressive effect of PRIMA-1Met/APR-246 was markedly reduced in SCLC cell lines transfected with p53 siRNA, supporting the role of mutant p53 in PRIMA-1Met/APR-246-induced cell death. Moreover, in vivo studies showed significant antitumor effects of PRIMA-1Met after i.v. injection in SCLC mouse models with no apparent toxicity. Conclusion: This study is the first to show the potential use of p53-reactivating molecules such as PRIMA-1Met/APR-246 for the treatment of SCLC. Clin Cancer Res; 17(9); 2830–41. ©2011 AACR.

Small Cell Lung Cancer: Can Recent Advances in Biology and Molecular Biology Be Translated into Improved Outcomes?

Paul A. Bunn Jr., MD, John D. Minna, MD, Alexander Augustyn, PhD, Adi F. Gazdar, MD, Youcef Ouadah, BS, Mark A. Krasnow, MD, PhD, Anton Berns, PhD, Elisabeth Brambilla, MD, Natasha Rekhtman, MD, PhD, Pierre P. Massion, MD, Matthew Niederst, PhD, Martin Peifer, PhD, Jun Yokota, MD, Ramaswamy Govindan, MD, John T. Poirier, PhD, Lauren A. Byers, MD, Murry W. Wynes, PhD, David G. McFadden, MD, PhD, David MacPherson, PhD, Christine L. Hann, MD, PhD, Anna F. Farago, MD, PhD, Caroline Dive, PhD, Beverly A. Teicher, PhD, Craig D. Peacock, PhD, Jane E. Johnson, PhD, Melanie H. Cobb, PhD, Hans-Guido Wendel, MD, David Spigel, MD, Julien Sage, PhD, Ping Yang, MD, PhD, M. Catherine Pietanza, MD, Lee M. Krug, MD, John Heymach, MD, PhD, Peter Ujhazy, MD, PhD, Caicun Zhou, MD, PhD, Koichi Goto, MD, Afshin Dowlati, MD, Camilla Laulund Christensen, PhD, Keunchil Park, MD, PhD, Lawrence H. Einhorn, MD, Martin J. Edelman, MD, Giuseppe Giaccone, MD, PhD, David E. Gerber, MD, Ravi Salgia, MD, PhD, Taofeek Owonikoko, MD, PhD, Shakun Malik, MD, Niki Karachaliou, MD, David R. Gandara, MD, Ben J. Slotman, MD, PhD, Fiona Blackhall, MD, PhD, Glenwood Goss, MD, FRCPC, Roman Thomas, MD, Charles M. Rudin, MD, PhD, Fred R. Hirsch, MD, PhD*

D-2-hydroxyglutarate produced by mutant IDH2 causes cardiomyopathy and neurodegeneration in mice

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) have been discovered in several cancer types and cause the neurometabolic syndrome D2-hydroxyglutaric aciduria (D2HGA). The mutant enzymes exhibit neomorphic activity resulting in production of D2-hydroxyglutaric acid (D-2HG). To study the pathophysiological consequences of the accumulation of D-2HG, we generated transgenic mice with conditionally activated IDH2(R140Q) and IDH2(R172K) alleles. Global induction of mutant IDH2 expression in adults resulted in dilated cardiomyopathy, white matter abnormalities throughout the central nervous system (CNS), and muscular dystrophy. Embryonic activation of mutant IDH2 resulted in more pronounced phenotypes, including runting, hydrocephalus, and shortened life span, recapitulating the abnormalities observed in D2HGA patients. The diseased hearts exhibited mitochondrial damage and glycogen accumulation with a concordant up-regulation of genes involved in glycogen biosynthesis. Notably, mild cardiac hypertrophy was also observed in nude mice implanted with IDH2(R140Q)-expressing xenografts, suggesting that 2HG may potentially act in a paracrine fashion. Finally, we show that silencing of IDH2(R140Q) in mice with an inducible transgene restores heart function by lowering 2HG levels. Together, these findings indicate that inhibitors of mutant IDH2 may be beneficial in the treatment of D2HGA and suggest that 2HG produced by IDH mutant tumors has the potential to provoke a paraneoplastic condition.

Synergy of radiotherapy and PD-1 blockade in Kras-mutant lung cancer

Radiation therapy (RT), a critical modality in the treatment of lung cancer, induces direct tumor cell death and augments tumor-specific immunity. However, despite initial tumor control, most patients suffer from locoregional relapse and/or metastatic disease following RT. The use of immunotherapy in non-small-cell lung cancer (NSCLC) could potentially change this outcome by enhancing the effects of RT. Here, we report significant (up to 70% volume reduction of the target lesion) and durable (up to 12 weeks) tumor regressions in conditional Kras-driven genetically engineered mouse models (GEMMs) of NSCLC treated with radiotherapy and a programmed cell death 1 antibody (αPD-1). However, while αPD-1 therapy was beneficial when combined with RT in radiation-naive tumors, αPD-1 therapy had no antineoplastic efficacy in RT-relapsed tumors and further induced T cell inhibitory markers in this setting. Furthermore, there was differential efficacy of αPD-1 plus RT among Kras-driven GEMMs, with additional loss of the tumor suppressor serine/threonine kinase 11/liver kinase B1 (Stk11/Lkb1) resulting in no synergistic efficacy. Taken together, our data provide evidence for a close interaction among RT, T cells, and the PD-1/PD-L1 axis and underscore the rationale for clinical combinatorial therapy with immune modulators and radiotherapy.

Targeted Cytosine Deaminase-Uracil Phosphoribosyl Transferase Suicide Gene Therapy Induces Small Cell Lung Cancer–Specific Cytotoxicity and Tumor Growth Delay

Purpose: Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment. Novel therapies are therefore in great demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug. Experimental design: The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter insulinoma-associated 1 (INSM1). Therapeutic effect was evaluated in vitro in SCLC cell lines and in vivo in SCLC xenografted nude mice using the nonviral nanoparticle DOTAP/cholesterol for transgene delivery. Results: INSM1-YCD/5-FC and INSM1-YCD-YUPRT/5-FC therapy induced high cytotoxicity in a range of SCLC cell lines. The highest therapeutic effect was obtained from the YCD-YUPRT fusion gene strategy. No cytotoxicity was induced after treatment of cell lines of other origin than SCLC. In addition the INSM1-YCD-YUPRT/5-FC therapy was superior to an established suicide gene system consisting of the herpes simplex virus thymidine kinase (HSVTK) gene and the prodrug ganciclovir. The superior effect was in part due to massive bystander cytotoxicity of YCD-YUPRT-produced toxins. Finally, INSM1-YCD-YUPRT/5-FC therapy induced significant tumor growth delay in SCLC xenografts compared with control-treated xenografts. Conclusions: The current study is the first to test cytosine deaminase-based suicide gene therapy for SCLC and the first to show an antitumor effect from the delivery of suicide gene therapeutics for SCLC in vivo. Clin Cancer Res; 16(8); 2308–19. ©2010 AACR.

First-in-human uPAR PET: Imaging of Cancer Aggressiveness

A first-in-human clinical trial with Positron Emission Tomography (PET) imaging of the urokinase-type plasminogen activator receptor (uPAR) in patients with breast, prostate and bladder cancer, is described. uPAR is expressed in many types of human cancers and the expression is predictive of invasion, metastasis and indicates poor prognosis. uPAR PET imaging therefore holds promise to be a new and innovative method for improved cancer diagnosis, staging and individual risk stratification. The uPAR specific peptide AE105 was conjugated to the macrocyclic chelator DOTA and labeled with 64Cu for targeted molecular imaging with PET. The safety, pharmacokinetic, biodistribution profile and radiation dosimetry after a single intravenous dose of 64Cu-DOTA-AE105 were assessed by serial PET and computed tomography (CT) in 4 prostate, 3 breast and 3 bladder cancer patients. Safety assessment with laboratory blood screening tests was performed before and after PET ligand injection. In a subgroup of the patients, the in vivo stability of our targeted PET ligand was determined in collected blood and urine. No adverse or clinically detectable side effects in any of the 10 patients were found. The ligand exhibited good in vivo stability and fast clearance from plasma and tissue compartments by renal excretion. In addition, high uptake in both primary tumor lesions and lymph node metastases was seen and paralleled high uPAR expression in excised tumor tissue. Overall, this first-in-human study therefore provides promising evidence for safe use of 64Cu-DOTA-AE105 for uPAR PET imaging in cancer patients.

Image-guided radiotherapy platform using single nodule conditional lung cancer mouse models

Close resemblance of murine and human trials is essential to achieve the best predictive value of animal-based translational cancer research. Kras-driven genetically engineered mouse models of non-small-cell lung cancer faithfully predict the response of human lung cancers to systemic chemotherapy. Owing to development of multifocal disease, however, these models have not been usable in studies of outcomes following focal radiotherapy (RT). We report the development of a preclinical platform to deliver state-of-the-art image-guided RT in these models. Presence of a single tumour as usually diagnosed in patients is modelled by confined injection of adenoviral Cre recombinase. Furthermore, three-dimensional conformal planning and state-of-the-art image-guided dose delivery are performed as in humans. We evaluate treatment efficacies of two different radiation regimens and find that Kras-driven tumours can temporarily be stabilized upon RT, whereas additional loss of either Lkb1 or p53 renders these lesions less responsive to RT. Current genetic mouse models of lung cancer develop multifocal tumours in all lobes, which limits their applicability to model radiotherapy of human disease. Here Herter-Sprie et aldevelop a method to induce single lung tumours in these models, allowing precise evaluation of radiation regiment efficacy.