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 radioand 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. LIST OF SCIENTIFIC PAPERS The thesis is based on the following papers, hereby denoted with Roman numbers: I. Berglund E, Akçakaya P, Berglund D, Karlsson F, Vukojević V, Lee L, Bogdanović D, Lui WO, Larsson C, Zedenius J, Fröbom R, Bränström R. Functional role of the Ca-activated Cl channel DOG1/TMEM16A in gastrointestinal stromal tumor cells. Experimental Cell Research 2014;326(2):315-325. doi: 10.1016/j.yexcr. 2014.05. 003 II. Fröbom R, Sellberg F, Xu C, Zhao A, Larsson C, Lui WO, Nilsson IL, Berglund E, Bränström R. Biochemical inhibition of DOG1/TMEM16A achieves antitumoral effects in human gastrointestinal stromal tumor cells in vitro. Anticancer Research 2019;39(7):3433-3442. doi: 10.21873/anticanres.13489 III. Sellberg F*, Fröbom R*, Binder C, Berglund D, Berglund E. Carbazate-activated polyvinyl alcohol (PVAC) as an antitumoral polymer. Manuscript. IV. Fröbom R, Berglund E, Aspinwall CA, Lui WO, Nilsson IL, Larsson C, Bränström R. Direct inhibition of the ATP-sensitive K channel by tyrosine kinase inhibitors imatinib, sunitinib and nilotinib. Manuscript. V. Fröbom R*, Berglund E*, Berglund D, Nilsson IL, Åhlén J, Von Sivers K, Linder Stragliotto C, Suenaert P, Karlsson-Parra A, Bränström R. Phase 1 trial evaluating safety and efficacy of intratumorally administered inflammatory allogeneic dendritic cells (ilixadencel) in advanced gastrointestinal stromal tumors. Manuscript. * Shared first authorship The published articles were printed with permission from the respective publisher.