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Targeting malignant B cells by manipulating expression of MCL-1 and NOXA

Tuesday 5 January 2016

Project
One of the hallmarks of cancer is the ability of cancer cells to resist cell death. Pro-survival B cell lymphoma 2 (BCL-2) family proteins are potent inhibitors of programmed cell death and are often over-expressed in cancer cells. MCL-1 is a potent cell death inhibitor and is overexpressed in many germinal center (GC) B cell-derived malignancies, such as follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL) and multiple myeloma (MM), and inhibiting MCL-1 expression in these malignant cells has been shown to result in rapid cell death.
We have recently identified multiple druggable targets that control the expression of MCL-1 in malignant B cells. However, it is unclear what the exact roles of these drug targets are on cell survival and whether their inhibition displays synergistic effects with existing drugs, such as BCL-2-inhibitor ABT-199 (Venetoclax). The Master student will examine the effects on cell survival using a panel of cell lines, comprising DLBCL, FL, MM and Burkitt’s lymphoma. This part of the project includes cell culture and flow cytometry as a read-out. Western blot en real time quantitative PCR will be used to assess effects on expression of MCL-1 and its upstream mediators.
A second part of the project concerns the role of NOXA on MCL-1 expression. Multiple drugs that are currently used in the clinic are aimed at increasing availability of NOXA, such as proteasome inhibitor Bortezomib. However, these drugs do not specifically affect NOXA and reveal multiple side-effects. The aim of this part of the project is to investigate the impact of increased NOXA on malignant B cells. We will make use of fluorescently labeled synthetic NOXA protein that we will introduce into cells using streptolysin O (a bacterial alternative for perforin). The effects on cell survival, cellular localization and inhibition of MCL-1 will be assessed by fluorescence microscopy and flow cytometry.


Figure 1: The BCL-2 family

http://www.tumor-immunology-utrecht.nl/victor-peperzak-1-1/

Techniques
Molecular biology: real time qPCR, Western blot
Cell biology: cell culture, fluorescence microscopy, flow cytometry

Duration
6 or 9 months

Contact
Dr Victor Peperzak, tel. 088 75 673 91, v.peperzak@umcutrecht.nl
Dr Kristin Denzer, tel. 088 75 643 68, k.denzer@umcutrecht.nl