Camilla S. Knudsen

Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway

Cyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific β-glucosidases following tissue damage results in the release of hydrogen cyanide. The cyanogenesis deficient1 (cyd1) mutant of Lotus japonicus carries a partial deletion of the CYP79D3 gene, which encodes a cytochrome P450 enzyme that is responsible for the first step in cyanogenic glucoside biosynthesis. The genomic region surrounding CYP79D3 contains genes encoding the CYP736A2 protein and the UDP-glycosyltransferase UGT85K3. In combination with CYP79D3, these genes encode the enzymes that constitute the entire pathway for cyanogenic glucoside biosynthesis. The biosynthetic genes for cyanogenic glucoside biosynthesis are also co-localized in cassava (Manihot esculenta) and sorghum (Sorghum bicolor), but the three gene clusters show no other similarities. Although the individual enzymes encoded by the biosynthetic genes in these three plant species are related, they are not necessarily orthologous. The independent evolution of cyanogenic glucoside biosynthesis in several higher plant lineages by the repeated recruitment of members from similar gene families, such as the CYP79s, is a likely scenario.

Characterization of a dynamic metabolon producing the defense compound dhurrin in sorghum

Metabolite channeling by a dynamic metabolon The specialized metabolite dhurrin breaks down into cyanide when plant cell walls have been chewed, deterring insect pests. Laursen et al. found that the enzymes that synthesize dhurrin in sorghum assemble as a metabolon in lipid membranes (see the Perspective by Dsatmaichi and Facchini). The dynamic nature of metabolon assembly and disassembly provides dhurrin on an as-needed basis. Membrane-anchored cytochrome P450s cooperated with a soluble glucosyltransferase to channel intermediates toward efficient dhurrin production. Science, this issue p. 890; see also p. 829 Enzymes that synthesize a specialized metabolite congregate and disperse on an as-needed basis in the lipid membrane. Metabolic highways may be orchestrated by the assembly of sequential enzymes into protein complexes, or metabolons, to facilitate efficient channeling of intermediates and to prevent undesired metabolic cross-talk while maintaining metabolic flexibility. Here we report the isolation of the dynamic metabolon that catalyzes the formation of the cyanogenic glucoside dhurrin, a defense compound produced in sorghum plants. The metabolon was reconstituted in liposomes, which demonstrated the importance of membrane surface charge and the presence of the glucosyltransferase for metabolic channeling. We used in planta fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to study functional and structural characteristics of the metabolon. Understanding the regulation of biosynthetic metabolons offers opportunities to optimize synthetic biology approaches for efficient production of high-value products in heterologous hosts.

Dynamic metabolic solutions to the sessile life style of plants

Plants are sessile organisms. To compensate for not being able to escape when challenged by unfavorable growth conditions, pests or herbivores, plants have perfected their metabolic plasticity by having developed the capacity for on demand dynamic biosynthesis and storage of a plethora of phytochemicals.

Tissue factor targeted radionuclide therapy with 177Lu-FVIIai inhibits tumor growth of human pancreatic cancer xenografts

Introduction: Tissue factor (TF) is involved in cancer processes responsible for aggressiveness and invasiveness and there is a correlation between tumor TF expression, metastatic potential, and patient outcome. The aim of the study was to develop a novel compound for localized TF targeted radionuclide therapy based on Factor VII (FVII), the natural ligand to TF. In the current study, we investigated the biodistribution and therapeutic potential of 177Lu labeled active site inhibited FVIIa (177Lu-FVIIai) for radionuclide therapy targeting TF in an experimental mouse model of pancreatic cancer. Methods: p-SCN-Bn-CHX-A’’-DTPA was conjugated to FVIIai followed by radiolabeling with 177Lu (177Lu-CHX-A’’-DTPA-FVIIai). A pancreas xenograft mouse model (BxPC3) was used to assess the therapeutic potential of 177Lu-FVIIai. NMRI nude mice were injected subcutaneously with BxPC3 cells. When tumors reached 50 mm3, the mice were randomized into groups receiving 177Lu-FVIIai, FVIIai, or vehicle. 177Lu-FVIIai was administered in doses of 15 MBq, 7.5 MBq or 2 x 7.5 MBq (n=8 mice/group). Tumor growth was monitored by 3 weekly measurements. Ex vivo biodistribution of 177Lu-FVIIai was studied in several organs at 1, 4, 24, 72 and 168 hours after injection. The in vivo biodistribution was evaluated by SPECT/CT imaging. Furthermore, competition and dose escalation experiments (1-30 MBq) were performed. Toxicity effects of the treatment with 177Lu-FVIIai were evaluated by hematology. Results: FVIIai was successfully radiolabeled with 177Lu with a high specific activity of 10-25 GBq/µmol after EDTA scavenging and PD-10 purification. No difference in tumor growth was observed between the FVIIai and vehicle groups. Mice receiving 15 MBq 177Lu-FVIIai had a significantly reduced tumor growth from day 0 to day 19 compared with mice from the control groups (425.5±44.8% versus 614.2±49.1%; p=0.02). No significant difference in tumor growth was observed in the groups receiving 7.5 MBq or 2 x 7.5 MBq compared with controls on day 19. There was a significant increase in the survival of mice treated with 7.5 MBq 177Lu-FVIIai compared with the controls (p=0.007). Treatment with 15 and 2 x 7.5 MBq 177Lu-FVIIai did not influence survival. Ex vivo tumor uptake of 177Lu-FVIIai was 1.16±0.04, 1.97±0.18, 1.95±0.07, 1.01±0.06, 0.31±0.02 percent injected dose per gram (%ID/g) at 1, 4, 24, 72 and 168 hours post-injection, respectively. Competition with unlabeled FVIIai 10 minutes before 177Lu-FVIIai injection significantly reduced tumor uptake of 177Lu-FVIIai (from 2.5±0.16 %ID/g to 1.7±0.05 %ID/g; p Conclusion: FVIIai was successfully radiolabeled with 177Lu. 177Lu-FVIIai showed therapeutic potential in a mouse model of human pancreatic cancer. Citation Format: Mette Munk Jensen, Jesper Fonslet, Camilla S. Knudsen, Troels E. Jeppesen, Andreas I. Jensen, Gregory W. Severin, Carsten H. Nielsen, Andreas Kjaer. Tissue factor targeted radionuclide therapy with 177Lu-FVIIai inhibits tumor growth of human pancreatic cancer xenografts [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 5203. doi:10.1158/1538-7445.AM2017-5203

Abstract 2805: A panel of orthotopic glioblastoma multiforme (GBM) patient derived xenograft (PDX) mouse models for efficacy evaluation of drugs

Background: Patients with glioblastoma multiforme (GBM) have a poor prognosis and few treatment options; hence new treatments are needed. Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, orthotopic implantation confers a translational advantage as the cancer develops in a microenvironment more closely mimicking that of the original patient tumor. Also the major impact of the blood brain barrier that must be taking into account when targeting brain tumors as GBM in terms of drug bioavailability is better represented in the orthotopic models. The aim of this study was therefore to develop a panel of orthotopic GBM PDX models for pre-clinical efficacy studies of new drugs. The models were then used to study the efficacy of standard of care such as temozolomide (TMZ) and external radiation therapy (XRT). Methods: Low passage subcutaneous tumors from six different PDX GBM models designated ST108, ST112, ST146, ST545, ST610 and ST2473 were digested and used for intracranial stereotactic injection in nude mice. Tumor take and growth was determined by T2-weighted magnetic resonance imaging (MRI). At confirmed tumor take mice were either treated with TMZ (100mg/kg/day for 5 days) or whole brain XRT (2 Gy/day for 5 days). Control groups receiving vehicle or sham XRT were included depending on treatment regiment. Final endpoint was survival by humane endpoints and tumors were fixed in formalin for histological evaluation. Results: MRI confirmed tumor take in all models within 5 weeks of implantation. The take rate was > 80% across all models. TMZ showed efficacy in the orthotopic ST610 GBM PDX model evaluated by MRI on day 14 (16.2±2.9 mm3 vs. 76.8±13.1 mm3, p=0.016), whereas the ST146 model displayed resistance to TMZ on day 14 (12.7±5.6 mm3 vs. 26.5±11.9 mm3, p=0.26). The median survival was 60 days vs. 14 days in the ST610 model (TMZ vs. vehicle, p=0.0005) and 27 days vs. 13 days in the ST146 model (TMZ vs. vehicle, p=0.007). XRT showed efficacy in the orthotopic ST2473 model. Tumor volume was significantly smaller in treated vs. sham animals 11 days after inclusion (6.9±1.4 mm3 vs. 28.9±3.3 mm3, p=0.001). Also, a survival benefit was observed in XRT treated animals compared to sham. Histology confirmed the presence of orthotopic tumors and typical GBM pathology characteristics such as pseudopalisading tumor cells surrounding necrosis and micro vascular proliferation were identified. Conclusion: Six different orthotopic GBM PDX models were established from low passage subcutaneous PDX models. Models sensitive and resistant to TMZ were identified and histological GBM characteristics were identified. Together, the established panel of orthotopic PDX models can be used as a relevant translational platform for testing of new drugs in a setting that more closely mimics the GBM tumor microenvironment and the impact of the blood brain barrier in patients. Citation Format: Mette M. Jensen, Camilla S. Knudsen, Lotte K. Kristensen, Mette K. Nedergaard, Michael J. Wick, Kyriakos P. Papadopoulos, Anthony W. Tolcher, Andreas Kjaer, Carsten H. Nielsen. A panel of orthotopic glioblastoma multiforme (GBM) patient derived xenograft (PDX) mouse models for efficacy evaluation of drugs [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 2805. doi:10.1158/1538-7445.AM2017-2805

Abstract 5187: PET imaging of trastuzumab emtansine (T-DM1) drug delivery to intracranial patient derived xenograft (PDX) models of breast cancer metastasis

Background: It is estimated that 10-30% of all breast cancer patients at some point develop brain metastases. Overexpression of the human epidermal growth factor receptor 2 (HER2) is a independent risk factor for development of brain metastases. Up to 37% of patients with HER2-positive metastatic breast cancer develop brain metastases and half of these patients die as a result of failure to control the intracranial disease. A reason for this is the challenge to obtain efficient drug delivery across the blood brain barrier (BBB). Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, when targeting brain tumors or metastases, the major impact of the BBB on drug bioavailability must be taken into consideration. Clinical PET imaging with 64Cu or 89Zr labeled trastuzumab has previously been able to visualize breast cancer brain metastases. The aim of our study was to investigate if PET imaging with 64Cu-labeled trastuzumab was predictive of the efficacy of trastuzumab emtansine (T-DM1) in a HER2 positive breast cancer PDX model established as an intracranial brain metastases model. Methods: The intracranial PDX model was established by stereotactic intracranial injection of enzymatically digested ST1339 tumor tissue. At confirmed tumor take, mice were randomized into two arms: control and T-DM1 (10 mg/kg/week x4). Treatment response was monitored by contrast-enhanced T1- and T2-weighted Magnetic Resonance Imaging (MRI) and positron emission tomography (PET) with the amino acid radiotracer O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET). PET/CT imaging with 64Cu-trastuzumab was performed in animals with confirmed intracranial ST1339 tumors prior to treatment with T-DM1 (10 mg/kg/week x4). Results: T-DM1 treatment with 10 mg/kg/week x4 of mice with intracranial tumors inhibited tumor growth and prolonged survival compared to non-treated animals. A variable response within the treatment group was observed. Forty percent had tumor shrinkage while 60% exhibited tumor growth within the duration of the therapy. The intracranial tumors were clearly visible on the 64Cu-trastuzumab PET images co-registered with T2-weighted MR images for anatomical localization. Interestingly, the intracranial tumor uptake between the animals was rather heterogeneous. Conclusion: A treatment response to T-DM1 was observed in an intracranial ST1339 HER2 positive breast cancer PDX model. PET imaging with 64Cu-trastuzumab confirmed delivery of trastuzumab to the tumors. Further quantitative image analysis of the intracranial 64Cu-trastuzumab tumor uptake will reveal if the drug delivery measured by 64Cu-trastuzumab PET imaging is predictive of the efficacy of T-DM1 in a HER2 positive PDX breast cancer brain metastases model. Citation Format: Carsten Haagen Nielsen, Mette K. Nedergaard, Lotte K. Kristensen, Camilla S. Knudsen, Michael J. Wick, Kyri Papadopoulos, Anthony Tolcher, Andreas Kjaer. PET imaging of trastuzumab emtansine (T-DM1) drug delivery to intracranial patient derived xenograft (PDX) models of breast cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5187.

Abstract A15: Efficacy of trastuzumab emtansine (T-DM1) in subcutaneous and intracranial patient derived xenograft models of breast cancer metastasis

Background: It is estimated that 10-30% of all breast cancer patients at some point develop brain metastases. Overexpression of the human epidermal growth factor receptor 2 (HER2) is a independent risk factor for development of brain metastases. Up to 37% of patients with HER2-positive metastatic breast cancer develop brain metastases and half of these patients die as a result of failure to control the intracranial disease. A reason for this is the challenge of efficient drug delivery across the blood brain barrier (BBB). Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, when targeting brain tumors or metastases, the major impact of the BBB on drug bioavailability must be taken into consideration. The aim of this study was therefore to compare the efficacy of trastuzumab emtansine (T-DM1) in a HER2 positive breast cancer PDX model established subcutaneously and as an intracranial brain metastases model. Methods: Mice were implanted subcutaneously with the HER2 positive breast cancer PDX model designated ST1339 and randomized into 3 treatment arms: Control, T-DM1 (5 mg/kg/week x4) and T-DM1 (10 mg/kg/week x4). Treatment response of subcutaneous tumors was monitored by caliper measurements. The intracranial PDX model was established by stereotactic intracranial injection of enzymatically digested ST1339 tumor tissue. At confirmed tumor take, mice were randomized into two arms: Control and T-DM1 (10 mg/kg/week x4). Treatment response was monitored by contrast-enhanced T1- and T2-weighted Magnetic Resonance Imaging (MRI) and positron emission tomography (PET) with the amino acid radiotracer O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET). Results: T-DM1 at 10 mg/kg/week x4 effectively inhibited tumor growth in the subcutaneous model whereas treatment with 5 mg/kg/week x4 did not have any effect on tumor growth. T-DM1 treatment at 10 mg/kg/week x4 of mice with intracranial tumors inhibited tumor growth and prolonged survival compared to non-treated animals. Conclusion: A treatment response to T-DM1 was observed in both the subcutaneous and intracranial ST1339 HER2 positive breast cancer PDX model. With the combination of subcutaneous and intracranial PDX models of breast cancer and breast cancer brain metastases new drugs can thus be tested in preclinical models that more closely mimic the microenvironment and the challenges of drug delivery across the BBB in patients. Citation Format: Carsten H. Nielsen, Mette K. Nedergaard, Lotte K. Kristensen, Camilla S. Knudsen, Michael J. Wick, Kyri Papadopoulos, Anthony W. Tolcher, Andreas Kjaer. Efficacy of trastuzumab emtansine (T-DM1) in subcutaneous and intracranial patient derived xenograft models of breast cancer metastasis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A15.