10th German Pharm-Tox Summit / 91. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (DGPT)

Chair

Mechanisms of T-Cell Lymphomas as Cancers of the Immune System

Speaker

Lecture

The of role of the Unfolded Protein Response for therapy resistance in pancreatic cancer

Prof. Dr. Dr.

Epigenetic factors in the TKI-resistance of CML cells: the histone methyltransferase KMT2D

Cancer pharmacology

AP03

Introduction: Tyrosine kinase inhibitors (TKIs) provide an effective treatment of chronic myeloid leukemia (CML) by targeting its main cause, the BCR::ABL1 fusion protein. However, 25% of patients receiving TKIs develop resistance within the first five years of treatment. In an in vitro-TKI-resistance cell model, a variant of the histone methyltransferase KMT2D was acquired in imatinib-resistant CML cells. KMT2D, an enzyme involved in the methylation of histone H3K4, is clinically known for its association with Kabuki syndrome and cancer development.Objectives: Our research was designed to determine the role of epigenetic factors contributing to TKI-resistance in CML cells, particularly the influence of the histone methyltransferase KMT2D and its recurrent variant c.571C>T, p.(Arg191Trp/R191W).Methods: The presence of the KMT2D variant was validated in the imatinib-resistant cells using in-depth-sequencing. The methylated histone H3 was detected using immunoblotting and mRNA levels were quantified using qPCR. Transient transfections were performed with siRNA or KMT2D-encoding plasmids in K-562 CML cells followed by the assessment of cell fitness through analysis of cell number, viability and proliferation after treatment with 2 µM imatinib. Native cells transfected with the KMT2D variant were further treated with a lysine-specific demethylase 1 (LSD1)-inhibitor.Results: On mRNA level, KMT2D expression was downregulated in the imatinib-resistant cells harboring the variant p.(R191W) (IM-R3: -33.3%, p=0.03; IM-R7: -33.9%, p<0.001). In native CML cells, siRNA-mediated downregulation of KMT2D reduced the susceptibility to imatinib (proliferation: 2.0-fold, p = 0.01). Furthermore, the imatinib response of the resistant cells harboring the variant was reinforced after rescue of KMT2D expression as shown by reduced metabolic activity: IM-R3: -12.9% (p=0.02); IM-R7: -15.7% (p=0.02). KMT2D p.(R191W) cells exhibited a decreased response to imatinib (cell number: 2.0-fold, p<0.001). Lastly, inhibition of histone H3 demethylation by LSD1 was able to partially reestablish the susceptibility to imatinib in cells transfected with the variant (metabolic activity: -58.4%, p=0.007).Conclusion: The results indicate that KMT2D p.(R191W) is a loss-of-function mutation and that the decrease in KMT2D expression in CML cells promotes imatinib-resistance. This suggests the histone methyltransferase KMT2D as an interesting target in the efforts of overcoming TKI-resistance.

Applied Pharmacology

Short talk

Prof. Dr.

Organoid cultures of colon carcinoma as a 3D model for the investigation of cytostatic drug resistance

IntroductionColorectal cancer (CRC) is the second most common tumor disease in Germany, highlighting the need for optimized therapies and development of efficient and reliable test models. 3D cell models have shown to be better predictors for in vitro research than standard 2D models. Implementing those 3D cell systems into primary cell cultures isolated directly from patient samples offer even further insights. This methodology allows focusing on factors altering cancer cell proliferation and migration, correlating co-players such as cellular adhesion molecules and the bioactive signal lipid Sphingosine-1-phosphate (S1P).ObjectiveThe development of a well-developed in vitro system, derived from patient samples, is the initial aim of this project. Including the establishment of multiple spheroid and organoid cultures for cancer research, allowing a deeper insight into the mechanisms of cytostatic drug resistance in CRC, based on interaction of adhesion molecules and S1P. Materials and MethodsPrimary cells were derived from colon tissue and colon cancer tissue of CRC patients and used for in vitro analysis. 3D cell culture was implemented by a forced-floating method to generate spheroids and cultured in Matrigel for organoid formation. Human permanent cell line HCT116 was incubated with Staurosporine (STS), 5-Fluorouracil (5-FU). Apoptosis and cell surface expression of the Intercellular Adhesion Molecule 1 (ICAM-1) were determined by flow cytometry.ResultsViable cells were successfully isolated from patient tissue samples. Organoids and spheroids were generated from freshly isolated primary cells in short term culture. Results in HCT116 cells, comparing 2D and 3D cell culture, showed reduced apoptosis in spheroid cell units compared to 2D monolayer cells, when treated with STS (0.3 µM, 1 µM for 2 h). In addition, we observed elevated levels of ICAM-1 expression in 3D cultures compared to monolayer CRC cultures. Incubation with STS (0.3 µM, 1 µM for 2 h) resulted in a significant loss of ICAM-1 expression in CRC cells cultured as monolayer, but not in the 3D cultured cells.ConclusionThe developed model allows stable cultivation of primary CRC cells in 3D culture. 3D growth may protect CRC cells from apoptosis-associated loss of ICAM-1. These 3D cell models may be suitable tools to determine differential response to cytostatic drug treatment in CRC cells.

Short talk 5
Mechanisms of drug resistance and tolerance in tumor therapy

Termin

Chair

Programm

Short talk 5Mechanisms of drug resistance and tolerance in tumor therapy

Termin

Chair

Programm

Short talk 5
Mechanisms of drug resistance and tolerance in tumor therapy