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NCT Junior Research Group Immune Checkpoint Regulation in Cancer

State of the art

Advances in cancer biology and immunology have paved the way to treating cancer by directing the immune system to target neoplastic cells. This new field of cancer immunotherapy has shown great promise as a treatment strategy in the clinic. As different routes were pursued to direct the immune system to fight malignancies, therapeutic targeting of cancer immune checkpoint molecules proved to be remarkably effective as it diminished cancer immune resistance.

The majority of these therapies benefit from the fact that many tumors are immunogenic and are infiltrated by cytotoxic T lymphocytes (CTLs) expressing T-cell receptors (TCRs) that bind to tumor antigens (Fig. 1). To maintain immune resistance, tumor cells have to develop strategies to inhibit these CTLs. This process involves many interacting partners on both the antigen-presenting cells (APCs) and the T-lymphocytes. Although some key players have been identified such as the protein PD-L1 (programmed cell death ligand 1) most molecular components and their cellular regulation remain elusive.

cancer-immunity cycle

Figure 1: The cancer-immunity cycle.
Neoplastic cells release tumor antigens (1) that are presented by APCs (2). They prime and activate T-cells in the lymph node (3) which traffic through the blood stream (4) and infiltrate the tumor site (5). The infiltrated T-cells recognize (6) and kill tumor cells (7).

Adapted from Immunity, 2013, 39 (1), 1-10

Understanding Immune Checkpoint Regulation

Our research group focusses on identifying novel ways to modulate immune checkpoint activity to reduce immune resistance in cancer. To achieve this goal, we utilize arrayed and pooled functional genomics screening by CRISPR/Cas9 or RNA Interference (Fig. 2), which enables us to systematically study regulatory mechanisms that maintain and enhance the expression and activation of immune checkpoint components in a wide range of cancer types and cell lines. This in turn sheds light on the molecular basis of cancer cell immune resistance opening new possibilities for immunotherapy of cancer.

Functional genomics screening in cancer cells

Figure 2: Functional genomics screening in cancer cells.
Target gene activity is reduced by CRISPR/Cas9 or RNAi in a reporter cell line expressing fluorescently labeled components of the immune checkpoint. Cells with altered reporter expression are separated by fluorescence-activated cell sorting (FACS). Target gene identity is determined by deep sequencing.

Copyright: Mirko Theis

Methodology

  • Arrayed and pooled CRISPR/Cas9-based functional genomics screening
  • Robotics and automated liquid handling in genome-scale
  • Large-scale RNA Interference screening by shRNA or esiRNA
  • Genome-editing for the generation of knock-out and knock-in cell lines
  • High-throughput expression analysis (Nanostring)
  • Proteomics, e.g. protein-protein interaction analysis, ChiP-Seq
  • Immunofluorescence staining / fluorescence microscopy
  • Bacterial culture and vector cloning
  • Molecular and cell biology methods, e.g. deep-sequencing, qPCR, Western Blot, fluorescence-activated cell sorting (FACS)

Team

Dr. Mirko Theis
Junior Group Leader
Phone: +49 (0)351 796 5590
E-mail: mirko.theis(at)tu-dresden.de

Dr. Sandeep Sreevalsan
Post-Doc
Phone: +49 (0)351 463 40284
E-mail: sandeep.sreevalsan(at)tu-dresden.de

2017

Toyoda, Y., Cattin, C., Stewart, M., Poser, I., Theis, M., Buchholz, F., Kurzchalia, T., Hyman, A., Muller, D. Genome-scale single-cell mechanical phenotyping reveals disease-related genes involved in mitotic rounding. Nature Communications, 2017; 8: 1266, 1.

2016

Gebler, C., Lohoff, T., Paszkowski-Rogacz, M., Mircetic, J., Chakraborty, D., Camgoz, A., Hamann, M., Theis, M., Thiede, C., Buchholz, F. Inactivation of Cancer Mutations Utilizing CRISPR/Cas9. J Natl Cancer Inst., 2016; 109 (1): 1-4.

2015

Ding, L., Paszkowski-Rogacz, M., Winzi, M., Chakraborty, D., Theis, M., Singh, S., Ciotta, G., Poser, I., Roguev, A., Chu, W.K., Choudhary, C., Mann, M., Stewart, A.F., Krogan, N., Buchholz, F. Systems Analyses Reveal Shared and Diverse Attributes of Oct4 Regulation in Pluripotent Cells. Cell Systems, 2015; 2 (1): 141-51. 

Wermke, M., Camgoz, A., Paszkowski-Rogacz, M., Thieme, S., von Bonin, M., Dahl, A., Platzbecker, U., Theis, M., Ehninger, G., Brenner, S., Bornhauser, M., Buchholz, F. RNAi profiling of primary human AML cells identifies ROCK1 as a therapeutic target and nominates Fasudil as an anti-leukemic drug. Blood, 2015; 125 (24): 3760-8.

Theis, M., Paszkowski-Rogacz, M., Weisswange, I., Chakraborty, D., Buchholz. F. Targeting Human Long Noncoding Transcripts by Endoribonuclease-Prepared siRNAs. J Biomol Screen. 2015; 20 (8): 1018-26.

Schmitt-Engel, C., Schultheis, D., Schwirz, J., Ströhlein, N., Troelenberg, N., Majumdar, U., Dao, V.A., Grossmann, D., Richter, T., Tech, M., Dönitz, J., Gerischer, L., Theis, M., Schild, I., Trauner, J., Koniszewski, N.D.B., Küster, E., Kittelmann, S., Hu, Y., Lehmann, S., Siemanowski, J., Ulrich, J., Kristen, A.P., Schröder, R., Morgenstern, B., Stanke, M., Buchhholz, F., Frasch, M., Roth, S., Wimmer, E.A., Schoppmeier, M., Klingler, M., and Bucher, G. The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology. Nature Communications, 2015; 6, 7822.