Translational investigations of novel and current antitumoral therapies in gastrointestinal stromal tumors

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma. Its incidence is around 10-15 per million person-years, translating into 150 new cases each year in Sweden. The molecular background for the absolute majority of GIST is characterized by gain-offunction mutations in KIT or PDGFRA genes, both encode receptor tyrosine kinases, allowing for targeted treatment with imatinib. This has revolutionized the treatment of GIST, which is inherently radio- and chemotherapy insensitive. However, durable remissions are uncommon relating to the development of resistance. The overall aim of the thesis was to explore novel and current treatments in GIST, as few treatment alternatives exist. In paper I, we examined the functional role of DOG1 protein, a diagnostic marker, in GIST. The protein is a calcium-activated chloride channel. We determined the expression of DOG1 and found a difference between imatinib-sensitive and imatinib-resistant cell lines with regards to subcellular localization. Electrophysiological registration confirmed the modulating ability of the DOG1 activator and inhibitor. Only modest effect was seen on proliferation, DOG1 inhibition induced a shift from early apoptotic to late apoptotic cells in the imatinib-resistant cell line. In paper II, we used a new potent inhibitor (CaCCinh-A01) of DOG1. We confirmed its inhibitory effect on chloride currents using patch-clamp technique. The cell viability was reduced. Furthermore, colony formation ability was markedly decreased after incubation with CaCCinh-A01. CaCCinh-A01 also led to a G1-cell cycle arrest, which was not seen with T16inh-A01 treatment. Therefore, paper I and II, confirms that DOG1 could potentially be a target for therapy. In paper III, we explored the antitumoral effects of a novel polymer-based therapy (PVAC). In vitro experiments revealed PVAC potently induced a population of non-viable cells, in a non-linear dose-response relationship. In vivo PVAC inhibited tumor growth in immunocompetent mice, and an increased CD3+ cell infiltration intratumorally was observed. In paper IV, we explored the commonly used tyrosine kinase inhibitors imatinib, sunitinib, and nilotinib possible interaction with ATP-binding sites, in which we used murine pancreatic β-cells as ATP-sensitive K+ (KATP) channel donors. By using patch-clamp technique, we showed that all three tyrosine kinase inhibitors decreased the channel activity. Further studies revealed an increased channel activity with imatinib in the presence of ATP and ADP. In paper V, the aim was to determine the safety and efficacy of intratumorally injected allogeneic pro-inflammatory dendritic cells (ilixadencel) in patients with advanced GIST and progression on tyrosine kinase inhibitors. The study showed an acceptable safety profile, and promising radiological response was observed in two out of six patients. To conclude, this translational thesis adds knowledge to new potential targets and novel antitumoral strategies, and increases our understanding of current treatment. Lastly, a clinical study found encouraging response in some patients and warrants further studies

Inward and outward currents of native and cloned K(ATP) channels (Kir6.2/SUR1) share single-channel kinetic properties

BACKGROUND: The ATP-sensitive K(+) (K(ATP)) channel is found in a variety of tissues extending from the heart and vascular smooth muscles to the endocrine pancreas and brain. Common to all K(ATP) channels is the pore-forming subunit Kir6.x, a member of the family of small inwardly rectifying K(+) channels, and the regulatory subunit sulfonylurea receptor (SURx). In insulin secreting β-cells in the endocrine part of the pancreas, where the channel is best studied, the K(ATP) channel consists of Kir6.2 and SUR1. Under physiological conditions, the K(ATP) channel current flow is outward at membrane potentials more positive than the K(+) equilibrium potential around −80 mV. However, K(ATP) channel kinetics have been extensively investigated for inward currents and the single-channel kinetic model is based on this type of recording, whereas only a limited amount of work has focused on outward current kinetics. METHODS: We have estimated the kinetic properties of both native and cloned K(ATP) channels under varying ionic gradients and membrane potentials using the patch-clamp technique. RESULTS: Analyses of outward currents in K(ATP) and cloned Kir6.2ΔC26 channels, alone or co-expressed with SUR1, show openings that are not grouped in bursts as seen for inward currents. Burst duration for inward current corresponds well to open time for outward current. CONCLUSIONS: Outward K(ATP) channel currents are not grouped in bursts regardless of membrane potential, and channel open time for outward currents corresponds to burst duration for inward currents

Polyvinyl Alcohol Carbazate as a Polymer-Based Antitumoral Agent

Development of treatment resistance is a major concern during treatment of cancer, and there is an unmet need for therapeutic strategies with novel modes of action. Polyvinyl alcohol carbazate (PVAC) is a polymer compound with unique biological properties. Herein, we describe the antitumoral effects of PVAC. Three well-established cell lines GIST-T1, B16.F10, and A375 were used to determine the in vitro antitumoral effects of PVAC. Assessments included light microscopy, cell viability, cell cycle, and apoptosis assays. In vivo treatment safety and efficacy were characterized in one immunocompetent (B16.F10) mouse model and one athymic nude (MDA-MB-231) mouse model. Excised tumors were measured, weighed, stained for Ki-67, CD3, and histopathologically evaluated. Intact PVAC expressed a non-linear dose-response antitumoral effect in vitro, whereas its separate components, PVA and carbazate, did not display antitumoral effects alone. In vivo, PVAC induced a significant intratumoral CD3(+) T-cell recruitment in immunocompetent mice (B16.F10), which was associated with tumor growth inhibition. Although growth inhibition was not significant in athymic mice (MDA-MB-231), histopathological evaluation detected an increase in stromal tissue and leukocyte infiltration. In conclusion, we present evidence for PVAC antitumoral effects both in vitro and in vivo. The mode of action was not elucidated in vitro, but a potential mechanism of in vivo activity was observed, characterized by an increase of immune cells into both immunocompetent and athymic mice. This finding warrants further study to validate its possible role as an immunomodulatory polymeric agent.De två första författarna delar förstaförfattarskapet.</p

Direct interaction of the ATP-sensitive K+ channel by the tyrosine kinase inhibitors imatinib, sunitinib and nilotinib

The ATP-regulated K+ channel (KATP) plays an essential role in the control of many physiological processes, and contains a ATP-binding site. Tyrosine kinase inhibitors (TKI) are commonly used drugs, that primarily target ATP-binding sites in tyrosine kinases. Herein, we used the patch-clamp technique to examine the effects of three clinically established TKIs on KATP channel activity in isolated membrane patches, using a pancreatic β-cell line as a KATP channel source. In excised inside-out patches, the activity of the KATP channel was dose-dependently inhibited by imatinib with half-maximal concentration of approximately 9.4 μM. The blocking effect of imatinib was slow and reversible. No effect of imatinib was observed on either the large (KBK) or the small (KSK) conductance, Ca2+-regulated K+ channel. In the presence of ATP/ADP (ratio 1) addition of imatinib increased channel activity approximately 1.5-fold. Sunitinib and nilotinib were also found to decrease KATP channel activity. These findings are compatible with the view that TKIs, designed to interact at the ATP-binding pocket on the tyrosine receptor, also interact at the ATP-binding site on the KATP channel. Possibly, this might explain some of the side effects seen with TKIs.National Institute of Biomedical Imaging and BioengineeringOpen access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Phase I trial evaluating safety and efficacy of intratumorally administered inflammatory allogeneic dendritic cells (ilixadencel) in advanced gastrointestinal stromal tumors

Background The majority of patients with advanced gastrointestinal stromal tumor (GIST) develop resistance to imatinib, and subsequent treatments have limited efficacy. Ilixadencel (allogeneic inflammatory dendritic cells) is a cell-based immune primer injected intratumorally that previously has been clinically investigated in metastatic renal cell carcinoma and hepatocellular carcinoma. Methods The trial was a single arm phase I trial assessing safety and efficacy of ilixadencel in subjects with progressing advanced/metastatic GIST despite ongoing treatment with second or later lines of tyrosine kinase inhibitors (TKI). Three patients were progressing while on sunitinib (second line), one on regorafenib (third line), and two on pazopanib (fourth line). TKI treatment was maintained throughout, while two intratumoral injections of ilixadencel (10 × 106 viable and HLA-DR expressing cells per dose) were administered. Results No severe adverse events were found to be related to ilixadencel administration. Four patients showed continued tumor progression at 3 months per RECIST 1.1 and Choi criteria. One patient (on third line regorafenib) had stable disease for 9 months and another patient (on second line sunitinib) had stable disease at end of study (12 months) as per RECIST 1.1. These two patients developed a partial response as per Choi criteria with a duration of 3 and 6 months, respectively. The median progression-free survival (PFS) was 4.0 months. Conclusion Ilixadencel treatment presented an acceptable safety profile among advanced GIST patients who developed resistance to TKI. Encouraging radiological tumor responses were detected in 33% of treated patients, supporting further investigation

Secretome protein signature of human gastrointestinal stromal tumor cells

Strategies for correct diagnosis, treatment evaluation and recurrence prediction are important for the prognosis and mortality rates among cancer patients. In spite of major improvements in clinical management, gastrointestinal stromal tumors (GISTs) can still be deadly due to metastasis and recurrences, which confirms the unmet need of reliable follow-up modalities. Tumor-specific secreted, shed or leaked proteins (collectively known as secretome) are considered promising sources for biomarkers, and suitable for detection in biofluids. Herein, we stimulated cell secretion in the imatinib-sensitive GIST882 cell line and profiled the secretome, collected as conditioned media, by using a shotgun proteomics approach. We identified 764 proteins from all conditions combined, 51.3% being predicted as classically/non-classically secreted. The protein subsets found were dependent on the stimulatory condition. The significant increase in protein release by the classical pathway was strongly associated with markers already found in other cancer types. Furthermore, most of the released proteins were non-classically released and overlapped to a high degree with proteins of exosomal origin. Imatinib pre-treatment radically changed these secretory patterns, which can have clinical implications when investigating biomarkers in imatinib-treated versus non-treated GIST patients. Our results show, for the first time, that GISTs contain a secretome signature. In the search for suitable biomarkers in the more complex GIST patient samples, this study aids in the understanding of basic GIST secretome characteristics. •GIST cells contain a secretome signature.•The secretome consists of classically and non-classically released proteins.•The protein subsets are stimulatory-dependent.•The GIST secretome is strongly associated with proteins found in other cancers.•Imatinib pre-treatment radically change the GIST secretory patterns