Maria Rovithi

Response and toxicity prediction by MALDI‐TOF‐MS serum peptide profiling in patients with non‐small cell lung cancer

We validated a previously reported proteomic signature, associated with treatment outcome, in an independent cohort of patients with non‐small cell lung cancer (NSCLC). A novel peptide signature was developed to predict toxicity.

Development of bioluminescent chick chorioallantoic membrane (CAM) models for primary pancreatic cancer cells: a platform for drug testing

The aim of the present study was to develop chick-embryo chorioallantoic membrane (CAM) bioluminescent tumor models employing low passage cell cultures obtained from primary pancreatic ductal adenocarcinoma (PDAC) cells. Primary PDAC cells transduced with lentivirus expressing Firefly-luciferase (Fluc) were established and inoculated onto the CAM membrane, with >80% engraftment. Fluc signal reliably correlated with tumor growth. Tumor features were evaluated by immunohistochemistry and genetic analyses, including analysis of mutations and mRNA expression of PDAC pivotal genes, as well as microRNA (miRNA) profiling. These studies showed that CAM tumors had histopathological and genetic characteristic comparable to the original tumors. We subsequently tested the modulation of key miRNAs and the activity of gemcitabine and crizotinib on CAM tumors, showing that combination treatment resulted in 63% inhibition of tumor growth as compared to control (p < 0.01). These results were associated with reduced expression of miR-21 and increased expression of miR-155. Our study provides the first evidence that transduced primary PDAC cells can form tumors on the CAM, retaining several histopathological and (epi)genetic characteristics of original tumors. Moreover, our results support the use of these models for drug testing, providing insights on molecular mechanisms underlying antitumor activity of new drugs/combinations.

Insight in taste alterations during treatment with protein kinase inhibitors

The role of Protein Kinase Inhibitors (PKI) in the treatment of various types of cancer is increasingly prominent. Their clinical application is accompanied by the development of side effects, among which patient-reported taste alterations. These alterations are missed frequently, but impair nutritional intake, are associated with weight loss and often result in significant morbidity, especially in the context of chronic administration. Accurate reporting of taste alterations is hampered by lack of modules for symptom objectification and inadequate understanding on the underlying mechanisms. In this review we initially describe the physiology of taste and smell and the mechanism of action of PKIs. We proceed to summarize taste related side effects as reported in major clinical trials and describe possible causal factors. Lastly, an in-depth analysis is given on potential molecular pathways responsible for the PKI-induced taste alterations. Objectification of patient-reported symptoms and universal reporting, along with a better understanding of the underlying mechanisms, will lead to early recognition and optimized treatment, ultimately improving patient adherence and quality of life.

Pulsatile high-dose treatment with antiangiogenic tyrosine kinase inhibitors improves clinical antitumor activity

Angiogenesis comprises one of the core hallmarks of cancer, essential for tumor establishment and growth. Elucidating the components of the angiogenic molecular pathway led to the identification of the crucial signaling axis of vascular endothelial growth factor (VEGF). This facilitated the discovery and subsequent clinical integration of either monoclonal antibodies targeting the soluble form of VEGF, such as bevacizumab, or small molecules that block the intracellular kinase domain of VEGF receptor, thereby preventing downstream signaling. More specifically, the development of small molecule tyrosine kinase inhibitors (TKIs) was initially accompanied by two hypotheses, tightly intertwined; they exert their action exclusively on endothelial cells, not tumor cells, and consequently, since endothelial cells are genetically stable, these drugs are ‘‘resistant to resistance,’’ namely their efficacy is not limited by the development of resistance [1]. The drug development of antiangiogenic TKIs is mainly based on the concept that inhibition of angiogenesis induces tumor cell death indirectly, through substrate deprivation. Treatment strategies with TKIs are traditionally focused on continuous administration, some with built-in intervals to allow recovery from toxicities. The subsequent plateau in drug plasma concentration was hypothesized to induce continuous inhibition of angiogenesis via blockade of specific intracellular signaling. Proof of concept for this dogma is lacking, because it is currently impossible to determine inhibitory activity of these drugs in individual cells. In other words, no adequate and validated technique is available to assess drug-related inhibition of specific signaling cascades in the targeted endothelial cells in tumor samples from patients during treatment. TKIs are small, hydrophobic molecules that can easily cross the cellular membrane, enter the cell and interact with intracellular kinases. Their blocking capacity is dependent on their affinity for individual kinases [2]. Therefore, one may wonder how they could discriminate between different cell types and target one exclusively. In contrast, they most likely affect not only endothelial cells, but may direct their inhibitory effects on tumor cells and cells of the tumor microenvironment as well. In all these cells, the inhibitory activity of (antiangiogenic) TKIs is dependent on their discriminative affinity for specific kinases, the intracellular concentrations reached and the presence and activity of kinases in these cells. Based on some of these considerations, we studied the effect of multiple TKIs in vitro and found that they exhibit a direct tumoricidal effect on cancer cell lines in clinically relevant concentrations [3], as those measured in on-treatment tumor biopsies from mice and patients (Labots et al., unpublished data). Disassociating antiangiogenic TKIs from the concept of antiangiogenesisonly mediating function, these agents can potently inhibit tumor growth both in vitro and in vivo. Clinical application of treatment with antiangiogenic TKIs showed in multiple tumor types their potential to improve patient survival but simultaneously exposed their limitations. Failing expectations, intrinsic or, more commonly, acquired resistance remains a major hurdle and accounts for heterogeneous patient response to treatment [4]. & Henk M. W. Verheul h.verheul@vumc.nl

Abstract 2989: High-dose, intermittent sunitinib as an alternative treatment strategy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Increased exposure to sunitinib treatment is associated with improved outcome indicative of the importance of dose intensity in the efficacy/toxicity balance. The currently approved schedule (50 mg daily, 4 weeks on, 2 weeks off) precludes further dose intensification. We hypothesized that high doses of sunitinib would improve tumor response with limited toxicity, when applied intermittently. Methods: In vitro proliferation was studied with MTT assays, one week after 3-9 hrs exposure of a panel of cancer cell lines (786-0, HT29, MDA-MB231, HCC827, A431) to high (5 and 20uM) concentrations of tyrosine kinase inhibitors (TKIs), namely sunitinib, sorafenib, erlotinib, pazopanib and axitinib. Apo-ONETM Homogenous Caspase 3/7 Assay (Promega) was applied to detect activation of apoptosis. The chick embryo chorioallantoic membrane (CAM) in vivo model was employed to further test the efficacy of the new scheduling, where sunitinib was topically administered on HT29 tumors growing on the CAM either daily at the commonly used dose of 40 mg/kg or once weekly at 200 mg/kg. Microvessel density (MVD) was determined by CD31 immunohistochemical staining. Plasma sunitinib concentrations were determined by LC-MS-MS from refractory patients with solid tumors participating in a phase I clinical trial (classical 3+3 design, EudraCT No: 2012-005756-41) in which high-dose, intermittent sunitinib treatment is being studied. Results: Sunitinib was identified as one of the most potent inhibitors of cell growth from the panel of TKIs; 6 hrs exposure to 20 uM of sunitinib resulted in tumor cell death. This effect was tumor cell line-independent and mediated via activation of caspase 3/7 pathway. High dose treatment of CAM tumors resulted in significant decrease in tumor growth compared to the control (mean 0.17 mg vs. 0.23 mg respectively, p=0.04), irrespective of angiogenesis inhibition as denoted by lack of inhibitory effect on MVD. It also led to higher intratumoral sunitinib concentrations compared to the daily schedule (14.3 vs. 8 ng/mg tissue, respectively), while blood concentrations were identical between the two schedules, averaging at 170 ng/ml. Patients enrolled in the first cohort of the clinical trial received 200 mg sunitinib orally weekly. This schedule was well tolerated and no dose limiting toxicities occurred. Overall, sunitinib exhibited moderate interpatient variability. Maximum plasma concentrations on Day 1 averaged on 161 ng/ml (range 118-239.5 ng/ml) and were reached approximately 6 hours after administration. Plasma drug concentrations decreased to undetectable from one cycle to the other. We have proceeded to escalate the dose for the next cohort of patients. Conclusion: Optimization of therapy with targeted agents remains an unmet challenge. Thus far, high-dose, intermittent sunitinib is well tolerated. These findings open new avenues that might contribute to improved treatment with sunitinib. Citation Format: Maria Rovithi, Richard R. de Haas, Richard J. Honeywell, Johannes Voortman, Arjan W. Griffioen, Mariette Labots, Anne Marije Luik, Godefridus J. Peters, Henk J. Broxterman, Henk M.w. Verheul. High-dose, intermittent sunitinib as an alternative treatment strategy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2989. doi:10.1158/1538-7445.AM2014-2989

Abstract 1621: Short-time exposure to high concentrations of sunitinib causes tumor cell death.

Background: Sunitinib, although initially developed as an antiangiogenic agent, has been shown to exert direct anti-tumor effects. Previously we demonstrated that sunitinib inhibits proliferation of various tumor cell lines in a dose-dependent manner. We here studied whether short exposure to high peak concentrations of sunitinib would induce tumor cell death. Methods: 786-O renal cancer cells were incubated with increasing concentrations of sunitinib (5, 10 and 20 μM) for various time intervals (1 hour, 3, 6, 9, 24, 48, 72 hours). At prespecified time points, sunitinib was removed and cells were subsequently allowed to grow in drug-free culture medium. Viability of these cells one week after short time sunitinib exposure and sensitivity of recovered cells after repetitive treatment with high sunitinib concentrations were determined by MTT assays. Flow cytometry with Propidium Iodide staining was employed to determine apoptosis. Results: Short-time exposure to high concentrations of sunitinib significantly induced cell death. Exposure to 20 μM of sunitinib for 9 hours provoked cell death in a manner identical to the prolonged exposure of 24, 48 or 72 hours. Flow cytometry confirmed the significant increase in cell death (60%) after 9-hour treatment with 20 μM sunitinib, compared to the untreated control (2%). Tumor cells that were repetitively treated with high concentrations of sunitinib retain their sensitivity to the drug identical to its parental cell line. Analysis of the key signaling pathways involved in sunitinib-induced cell death is ongoing. Conclusion: We here report that repetitive short-time exposure to high concentration sunitinib causes cell death without inducing resistance. Interestingly, single-dose sunitinib up to 300 mg was previously reported to be well tolerated by patients. Based on these findings, administration of high dose intermittent sunitinib may result in cytotoxic antitumor activity irrespective of angiogenesis inhibition. A phase I trial to test this hypothesis in patients with cancer will start shortly. Citation Format: Maria Rovithi, Dennis Poel, Jens Voortman, Mariette Labots, Henk Dekker, Kristy J. Gotink, Henk J. Broxterman, Henk M.W. Verheul. Short-time exposure to high concentrations of sunitinib causes tumor cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1621. doi:10.1158/1538-7445.AM2013-1621

Abstract 3608: Measurement of kinase activity in cancer cell lines and tumor tissue using a tyrosine kinase peptide substrate array

Introduction Tyrosine kinases play an important role in tumor biology. Their activity can be measured using a kinase peptide substrate array consisting of 144 Tyr residue-containing peptides (PamChip®, PamGene, Den Bosch, The Netherlands). We evaluated this platform for the measurement of kinase activity in tumor tissue and cancer cell lines under various experimental conditions. Methods Lysates of colorectal and renal cancer cell lines, HCT116 and 786-0 respectively, were made using both Mammalian and Tissue Protein Extraction Reagent (M-PER and T-PER, Thermo Scientific) and Radio-Immunoprecipitation Assay (RIPA, home made) buffer. Lysates from patient-derived tumor tissues were prepared by adding T-PER to several 10 μm cryoslides containing >50% tumor. After lysate incubation with reaction buffer containing a fluorescent labeled antibody against phospo-tyrosine and ATP, kinase activity profiles were determined on kinetics of recorded peptide substrate phosphorylation intensities. The effect of protein and ATP concentration, different lysis buffers and number of freeze-thaw cycles on basal kinase activity was studied. Sunitinib, sorafenib and dasatinib, clinically available tyrosine kinase inhibitors (TKIs), were used to differentially inhibit kinase activity in the lysates. Results Application of 2.5-15 µg protein in 40 µl sample mix per array revealed linearly increasing phosphorylation signal intensities and initial velocities (Vini) of the kinetic curve (R2 = 0.98). Increasing ATP concentrations induced phosphorylation signal intensities, but above 400 µM the curve deviated from linearity. Basal kinase activity profiles of cell lines and tumor tissues were reproducible with CV9s below 15%, with good signal-to-background ratios and low aspecific binding. Different lysis buffers resulted in a maximum variation of phosphorylation signal intensity of 47±5.7% in both cell lines without affecting the actual profile. Quadruple freeze-thawing of lysates did not affect signal intensities by more than 10%. Inhibition profiles of treated vs. control lysates were reproducible within and between experiments, showing a higher and differential number of inhibited peptides at increasing TKI concentrations. In contrast to the ATP-independent inhibition of dasatinib, ATP-dependent inhibition for sunitinib and sorafenib was demonstrated by combining a fixed drug concentration with increasing concentrations of ATP up to 800 µM. Conclusion Kinase activity in lysates from cancer cell lines and patient-derived tumor tissue can be reproducibly profiled with a tyrosine kinase peptide substrate array. In addition, TKIs show differential ATP-dependent inhibition profiles on this array. Taken together, we expect that array-based tumor kinase activity profiling may lead to specific TKI-phosphorylation fingerprints for personalized treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3608. doi:1538-7445.AM2012-3608

Abstract 1374: Cross-resistance and sensitivity of sunitinib resistant tumor cells

Background: Resistance to sunitinib, a multi-targeted anti-angiogenic tyrosine kinase inhibitor (TKI) approved for clinical use in advanced renal cell cancer, gastrointestinal stromal tumors and pancreatic neuroendocrine tumors, represents a major clinical problem. Previously we have shown that tumor cells acquire resistance in vitro by continuous incubation with sunitinib and found that acidic lysosomal sequestration of sunitinib mediates its resistance. In this study, we investigated whether sunitinib resistant tumor cells are cross-resistant to other clinically relevant inhibitors and whether agents interfering with lysosomal function can modulate resistance. Methods: Continuous exposure to sunitinib for more than 12 months resulted in drug resistance in 786-O (RCC) and HT-29 (colorectal cancer) cell lines. Using these cells, cross-resistance to other clinically available TKIs such as sorafenib, pazopanib and erlotinib, and to the mTOR inhibitor everolimus, was determined in proliferation assays (MTT). In addition, modulation of sunitinib resistance by the lysosomal inhibitors (LIs) chloroquine and bafilomycin A1 was studied using proliferation assays. The effect of LIs on phosphorylation of key signaling proteins was determined by western blotting. Results: Compared to parental cell lines, proliferation assays revealed that sunitinib resistant cell lines were cross-resistant to pazopanib and erlotinib, while no significant decrease in sensitivity to sorafenib or everolimus was observed. Both chloroquine and bafilomycin A1 were able to modulate sunitinib resistance. Western blot analysis revealed that phosphorylation of key signaling proteins including Akt and p44/42-MAPK was inhibited in sunitinib resistant cells upon co-incubation with chloroquine, while sunitinib alone did not inhibit phosphorylation of these kinases. Conclusion: We found that sunitinib resistant tumor cells are cross-resistant to pazopanib and erlotinib, but remain sensitive to sorafenib and everolimus. Furthermore, combination with the lysosomal inhibitor chloroquine resulted in inhibition of key signaling proteins in sunitinib resistant cells. Taken together, we envision that these findings are relevant for clinical treatment strategies in patients with renal cell cancer resistant to sunitinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1374. doi:1538-7445.AM2012-1374