Karianne G. Fleten

Radiosensitization by the histone deacetylase inhibitor vorinostat under hypoxia and with capecitabine in experimental colorectal carcinoma

BackgroundThe histone deacetylase inhibitor vorinostat is a candidate radiosensitizer in locally advanced rectal cancer (LARC). Radiosensitivity is critically influenced by hypoxia; hence, it is important to evaluate the efficacy of potential radiosensitizers under variable tissue oxygenation. Since fluoropyrimidine-based chemoradiotherapy (CRT) is the only clinically validated regimen in LARC, efficacy in combination with this established regimen should be assessed in preclinical models before a candidate drug enters clinical trials.MethodsRadiosensitization by vorinostat under hypoxia was studied in four colorectal carcinoma cell lines and in one colorectal carcinoma xenograft model by analysis of clonogenic survival and tumor growth delay, respectively. Radiosensitizing effects of vorinostat in combination with capecitabine were assessed by evaluation of tumor growth delay in two colorectal carcinoma xenografts models.ResultsUnder hypoxia, radiosensitization by vorinostat was demonstrated in vitro in terms of decreased clonogenicity and in vivo as inhibition of tumor growth. Adding vorinostat to capecitabine-based CRT increased radiosensitivity of xenografts in terms of inhibited tumor growth.ConclusionsVorinostat sensitized colorectal carcinoma cells to radiation under hypoxia in vitro and in vivo and improved therapeutic efficacy in combination with capecitabine-based CRT in vivo. The results encourage implementation of vorinostat into CRT in LARC trials.

Combined inhibition of the cell cycle related proteins Wee1 and Chk1/2 induces synergistic anti-cancer effect in melanoma

BackgroundMalignant melanoma has an increasing incidence rate and the metastatic disease is notoriously resistant to standard chemotherapy. Loss of cell cycle checkpoints is frequently found in many cancer types and makes the cells reliant on compensatory mechanisms to control progression. This feature may be exploited in therapy, and kinases involved in checkpoint regulation, such as Wee1 and Chk1/2, have thus become attractive therapeutic targets.MethodsIn the present study we combined a Wee1 inhibitor (MK1775) with Chk1/2 inhibitor (AZD7762) in malignant melanoma cell lines grown in vitro (2D and 3D cultures) and in xenografts models.ResultsOur in vitro studies showed that combined inhibition of Wee1 and Chk1/2 synergistically decreased viability and increased apoptosis (cleavage of caspase 3 and PARP), which may be explained by accumulation of DNA-damage (increased expression of γ-H2A.X) - and premature mitosis of S-phase cells. Compared to either inhibitor used as single agents, combined treatment reduced spheroid growth and led to greater tumour growth inhibition in melanoma xenografts.ConclusionsThese data provide a rationale for further evaluation of the combination of Wee1 and Chk1/2 inhibitors in malignant melanoma.

Immunotoxin targeting EpCAM effectively inhibits peritoneal tumor growth in experimental models of mucinous peritoneal surface malignancies

Cytoreductive surgery and intraperitoneal (i.p.) chemotherapy constitute a curative treatment option in mucinous peritoneal surface malignancies of intestinal origin, but treatment outcome is highly variable and the search for novel therapies is warranted. Immunotoxins are attractive candidates for targeted therapy in the peritoneal cavity because of direct cytotoxicity, distinct mechanisms of action and tumor cell selectivity. The MOC31PE immunotoxin targets the tumor‐associated adhesion protein EpCAM (Epithelial Cell Adhesion Molecule), and has been administered safely in early clinical trials. In our work, the efficacy of i.p. administration of MOC31PE alone and together with mitomycin C (MMC) was investigated in unique animal models of human mucinous peritoneal surface malignancies. In initial model validation experiments, clear differences in efficacy were demonstrated between MMC and oxaliplatin, favoring MMC in five investigated tumor models. Subsequently, MOC31PE and MMC were given as single i.p. injections alone and in combination. In the PMCA‐2 model, moderate growth inhibition was obtained with both drugs, while the combination resulted in at least additive effects; whereas the PMP‐2 model was highly sensitive to both drugs separately and in combination and intermediate sensitivity was found for the PMCA‐3 model. Furthermore, results from ex vivo experiments on freshly obtained mucinous tumor tissue from animals and patients suggested that classic mechanisms of immunotoxin activity were involved, i.e., inhibition of protein synthesis and induction of apoptosis. The present results suggest that adding MOC31PE to MMC‐based i.p. chemotherapy should be further explored for EpCAM‐expressing peritoneal surface malignancies, and a phase I trial is in preparation.

Targeted cancer therapy with a novel anti-CD37 beta-particle emitting radioimmunoconjugate for treatment of non-hodgkin lymphoma

177Lu-DOTA-HH1 (177Lu-HH1) is a novel anti-CD37 radioimmunoconjugate developed to treat non-Hodgkin lymphoma. Mice with subcutaneous Ramos xenografts were treated with different activities of 177Lu-HH1, 177Lu-DOTA-rituximab (177Lu-rituximab) and non-specific 177Lu-DOTA-IgG1 (177Lu-IgG1) and therapeutic effect and toxicity of the treatment were monitored. Significant tumor growth delay and increased survival of mice were observed in mice treated with 530 MBq/kg 177Lu-HH1 as compared with mice treated with similar activities of 177Lu-rituximab or non-specific 177Lu-IgG1, 0.9% NaCl or unlabeled HH1. All mice injected with 530 MBq/kg of 177Lu-HH1 tolerated the treatment well. In contrast, 6 out of 10 mice treated with 530 MBq/kg 177Lu-rituximab experienced severe radiation toxicity. The retention of 177Lu-rituximab in organs of the mononuclear phagocyte system was longer than for 177Lu-HH1, which explains the higher toxicity observed in mice treated with 177Lu-rituximab. In vitro internalization studies showed that 177Lu-HH1 internalizes faster and to a higher extent than 177Lu-rituximab which might be the reason for the better therapeutic effect of 177Lu-HH1.

hvTRA, a novel TRAIL receptor agonist, induces apoptosis and sustained growth retardation in melanoma

In recent years, new treatment options for malignant melanoma patients have enhanced the overall survival for selected patients. Despite new hope, most melanoma patients still relapse with drug-resistant tumors or experience intrinsic resistance to the therapy. Therefore, novel treatment modalities beneficial for subgroups of patients are needed. TRAIL receptor agonists have been suggested as promising candidates for use in cancer treatment as they preferentially induce apoptosis in cancer cells. Unfortunately, the first generation of TRAIL receptor agonists showed poor clinical efficacy. hvTRA is a second-generation TRAIL receptor agonist with improved composition giving increased potency, and in the present study, we showed hvTRA-induced activation of apoptosis leading to an efficient and sustained reduction in melanoma cell growth in cell lines and xenograft models. Furthermore, the potential of hvTRA in a clinical setting was demonstrated by showing efficacy on tumor cells harvested from melanoma patients with lymph node metastasis in an ex vivo drug sensitivity assay. Inhibition of mutated BRAF has been shown to regulate proteins in the intrinsic apoptotic pathway, making the cells more susceptible for apoptosis induction. In an attempt to increase the efficacy of hvTRA, combination treatment with the mutated BRAF inhibitor vemurafenib was investigated. A synergistic effect by the combination was observed for several cell lines in vitro, and an initial cytotoxic effect was observed in vivo. Unfortunately, the initial increased reduction in tumor growth compared with hvTRA mono treatment was not sustained, and this was related to downregulation of the DR5 level by vemurafenib. Altogether, the presented data imply that hvTRA efficiently induce apoptosis and growth delay in melanoma models and patient material, and the potential of this TRAIL receptor agonist should be further evaluated for treatment of subgroups of melanoma patients.

Use of non-invasive imaging to monitor response to aflibercept treatment in murine models of colorectal cancer liver metastases.

The liver is the most frequent metastatic site in colorectal cancer (CRC), and relevant orthotopic in vivo models are needed to study the efficacy of anticancer drugs in the metastatic setting. A challenge when utilizing such models is monitoring tumor growth during the experiments. In this study, experimental liver metastases were established in nude mice by splenic injection of the CRC cell lines HT29 and HCT116, and the mice were treated with the antiangiogenic drug aflibercept. Tumor growth was monitored using magnetic resonance imaging (MRI) and bioluminescence imaging (BLI). Aflibercept treatment was well tolerated and resulted in increased animal survival in HCT116, but not in HT29, while inhibited tumor growth was observed in both models. Treatment efficacy was monitored with high precision using MRI, while BLI detected small-volume disease with high sensitivity, but was less accurate in end-stage disease. Apparent diffusion coefficient (ADC) values obtained by diffusion weighted MRI (DW-MRI) were highly predictive of treatment response, with increased ADC corresponding well with areas of necrosis observed by histological evaluation of aflibercept-treated xenografts. The results showed that the efficacy of the antiangiogenic drug aflibercept varied between the two models, possibly reflecting unique growth patterns in the liver that may be representative of human disease. Non-invasive imaging, especially MRI and DW-MRI, can be used to effectively monitor tumor growth and treatment response in orthotopic liver metastasis models.

Individual tumor volume responses to short-course oxaliplatin-containing induction chemotherapy in locally advanced rectal cancer – Targeting the tumor for radiation sensitivity?

BACKGROUND Neoadjuvant treatment of locally advanced rectal cancer (LARC) involves chemoradiotherapy (CRT), which may cause significant toxicity, and the potential role and sequential placement of neoadjuvant chemotherapy (NACT) relative to CRT is under debate. PATIENTS AND METHODS In a non-randomized study of 72 LARC patients, short-course oxaliplatin-containing NACT was administered prior to CRT. Tumor volumes were calculated from magnetic resonance images before and after NACT, and four weeks after CRT, and associations between tumor volume responses and outcome were analyzed. Additionally, the impact of oxaliplatin exposure on radiosensitivity was examined in colorectal carcinoma cell lines. RESULTS All tumors except one responded to NACT, with better responses in T3 than T4 cases, and 69/72 patients obtained additional tumor volume reduction after subsequent CRT. However, no associations were found between tumor volume reduction and long-term outcome. Of note, oxaliplatin-resistant cells were significantly more radiosensitive than the oxaliplatin-sensitive counterparts. CONCLUSIONS Oxaliplatin-containing NACT led to substantial tumor volume reduction with particularly good responses in T3 cases. NACT did not impede subsequent CRT response, and experimental results rather suggested enhanced radiosensitivity in oxaliplatin-exposed cells, encouraging studies to explore the administration of NACT prior to CRT. Data are still lacking to support omitting radiation in LARC management.

PET Brain Imaging of Neuropeptide Y2 Receptors Using N-11C-Methyl-JNJ-31020028 in Pigs

Neuropeptide Y2 (NPY2) receptors are implicated in diverse brain disorders, but no suitable PET radiotracers are currently available for studying NPY2 receptors in the living brain. We developed a novel positron-emitting radioligand based on the NPY2 receptor antagonist JNJ-31020028 (N-(4-(4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl)-3-fluorophenyl)-2-pyridin-3-ylbenzamide) and used the radiotracer for PET brain imaging in pigs. Methods: In vitro receptor autoradiography studies were performed to establish the anatomic distribution of NPY2 receptors in the pig brain. In vivo, baseline 90-min PET recordings of N-11C-methyl-JNJ-31020028 were conducted in anesthetized Yorkshire x Landrace pigs, concurrent with arterial blood sampling. Postchallenge scans were conducted after injection of unlabeled JNJ-31020028 as a pharmacologic intervention. Cyclosporine A was used to enhance levels of the PET radiotracer in the brain. The PET images were manually coregistered to a MR imaging atlas of the pig brain. Maps of the N-11C-methyl-JNJ-31020028 volume of distribution in the brain were prepared, and regional binding potentials of NPY2 receptors toward the radioligand were calculated using the simplified reference tissue method. Results: In autoradiography studies, N-11C-methyl-JNJ-31020028 receptor binding sites were observed primarily in the hippocampus and were inhibited by unlabeled JNJ-31020028. In PET studies, N-11C-methyl-JNJ-31020028 was metabolized slowly in the bloodstream, with 25% of the 11C-labeled parent compound remaining 30 min after injection. PET imaging showed baseline binding potentials of 0.64 ± 0.07 in the thalamus, 0.55 ± 0.02 in the caudate, and 0.49 ± 0.03 in the hippocampus. Graphical reference region analyses demonstrated that N-11C-methyl-JNJ-31020028 binding was reversible; infusion of unlabeled JNJ-31020028 markedly displaced the PET radioligand from binding sites in the hippocampus, thalamus, caudate nucleus, and cerebellum but not in the corpus callosum, which served as reference region for nonspecific binding. Conclusion: N-11C-methyl-JNJ-31020028 has several suitable properties for PET neuroimaging of NPY2 receptors. First, it is metabolized slowly in the bloodstream of pigs. Second, using cyclosporine, the target-to-background ratio of N-11C-methyl-JNJ-31020028 is sufficient for estimating pharmacokinetic parameters. Third, N-11C-methyl-JNJ-31020028 binds reversibly and competitively to cerebral sites known to contain relatively high numbers of NPY2 receptors, such as the hippocampus, thalamus, caudate nucleus, and cerebellum. Fourth, white matter such as corpus callosum, known to contain negligible numbers of NPY2 receptors, can serve as a reference region for estimating binding potentials in brain regions. To our knowledge, there is no other radioligand with these favorable properties and with this specificity for NPY2 receptors, which makes N-11C-methyl-JNJ-31020028 the first candidate radioligand for PET investigations of NPY2 receptors in the living brain.

MOC31PE immunotoxin – targeting peritoneal metastasis from epithelial ovarian cancer

Peritoneal metastasis (PM) is an important feature of epithelial ovarian cancer (EOC) and is a frequent site of drug resistant disease recurrence, identifying PM-EOC an important clinical challenge. The MOC31PE immunotoxin targets and kills tumor cells expressing the epithelial cell adhesion molecule (EpCAM), which is highly expressed in EOC, and MOC31PE is being investigated for use in treatment of PM-EOC. The efficacy of MOC31PE treatment alone and in combination with cytotoxic drugs was investigated in two human EpCAM expressing EOC cell lines, B76 and MDHA-2774, in vitro and in corresponding mouse models mimicking PM-EOC. MOC31PE efficaciously killed tumor cells alone and showed equal or superior activity in vitro (paclitaxel, cisplatin, carboplatin) and in vivo (paclitaxel, mitomycin C) compared to the investigated cytotoxic drugs. Additive, or importantly, no antagonistic effects were observed in combination experiments. In ex vivo cell culture, the cytotoxic effect of MOC31PE was studied on freshly isolated surgical EOC samples. All investigated fresh EOC samples expressed EpCAM and MOC31PE effectively reduced cell viability in ex vivo cultures. In conclusion, these results, together with our previous preclinical and clinical experience, support development of MOC31PE for treatment of PM-EOC in combination with currently used cytotoxic drugs.Peritoneal metastasis (PM) is an important feature of epithelial ovarian cancer (EOC) and is a frequent site of drug resistant disease recurrence, identifying PM-EOC an important clinical challenge. The MOC31PE immunotoxin targets and kills tumor cells expressing the epithelial cell adhesion molecule (EpCAM), which is highly expressed in EOC, and MOC31PE is being investigated for use in treatment of PM-EOC.The efficacy of MOC31PE treatment alone and in combination with cytotoxic drugs was investigated in two human EpCAM expressing EOC cell lines, B76 and MDHA-2774, in vitro and in corresponding mouse models mimicking PM-EOC. MOC31PE efficaciously killed tumor cells alone and showed equal or superior activity in vitro (paclitaxel, cisplatin, carboplatin) and in vivo (paclitaxel, mitomycin C) compared to the investigated cytotoxic drugs. Additive, or importantly, no antagonistic effects were observed in combination experiments.In ex vivo cell culture, the cytotoxic effect of MOC31PE was studied on freshly isolated surgical EOC samples. All investigated fresh EOC samples expressed EpCAM and MOC31PE effectively reduced cell viability in ex vivo cultures.In conclusion, these results, together with our previous preclinical and clinical experience, support development of MOC31PE for treatment of PM-EOC in combination with currently used cytotoxic drugs.

Abstract 927: Fibroblast-induced switching to the invasive phenotype and PI3K-mTOR signaling protects melanoma cells from BRAF inhibitors

Phenotypic heterogeneity of cancer cells can reason diversity in therapy responses within the same tumor, which might influence the overall efficacy of the treatment. Tumor stroma is an important contributor to intratumoral heterogeneity and can play a significant modulatory role in therapy response/resistance. Through studies on biological mechanisms of stroma-promoted resistance, novel targets could be identified for combination therapies aimed to eradicate both stroma-dependent and independent counterparts of the tumor. In this study we explore how the efficacy of the BRAF inhibitor (BRAFi) vemurafenib, a targeted agent commonly used against BRAF-mutant malignant melanoma, is modulated by stromal cells. By using multiple co-culture systems and experimental metastasis models, we showed that in the presence of lung fibroblasts, adjacent melanoma cells respond poorly to BRAFi. The protective influence of stroma was associated with stroma-induced changes in the melanoma cell phenotype, which was mapped by global gene expression and proteome analysis. We revealed that under the influence of fibroblasts, melanoma cells underwent a phenotype transition to the invasive, mesenchymal-like state characterized by down-regulation of melanocytic markers (MITF and its targets), up-regulation of receptor tyrosine kinases (RTKs)/RTK-linked signaling (like AXL or PDGFR activating down-stream PI3K) and elevation of extracellular-matrix fibronectin. We propose that these alterations allow melanoma cells to utilize alternative signaling pathways, like RTK-PI3K-mTOR instead of BRAF-driven MAPK, which reduces sensitivity to BRAFi. This scenario is further supported by the observations that: i) upon BRAFi treatment, stroma-protected melanoma maintained high levels of phospho-ribosomal protein S6 (pS6), a mTOR effector protein; ii) inhibition of mTOR or the upstream pathway PI3K together with BRAF eradicated pS6high subpopulations and enhanced the anti-proliferative effect in stroma-interacting melanoma; iii) the benefit of mTOR and BRAF co-inhibition was also seen in early-stage lung metastases in vivo. In conclusion, our findings signify the importance of stromal cells, specifically lung fibroblasts, in regulating melanoma cell phenotype and signaling, which impairs response to BRAFi. The stroma-induced invasive phenotype determinants that facilitate RTK-PI3K-mTOR signaling (e.g. AXL, PDGFR or fibronectin-binding integrins) could represent potential targets for overcoming stroma-mediated resistance to BRAFi. Currently, we are testing BRAFi in combination with several novel inhibitors of the identified RTKs and performing a cancer drug sensitivity screen to reveal the most effective drug combinations against stroma-interacting melanoma cells. Citation Format: Kotryna Seip, Karianne Giller Fleten, Anna Barkovskaya, Vigdis Nygaard, Mads Haugen Haugen, Birgit Ovstebo Engesaeter, Gunhild Mari Maelandsmo, Lina Prasmickaite. Fibroblast-induced switching to the invasive phenotype and PI3K-mTOR signaling protects melanoma cells from BRAF inhibitors. [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 927.