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Synthetic Lethal Interactions with nucleophosMin 1

Periodic Reporting for period 1 - SLIM (Synthetic Lethal Interactions with nucleophosMin 1)

Reporting period: 2016-07-01 to 2018-06-30

Original aim of SLIM was to study the effect of NPM1 mutations on the sensitivity of acute myeloid leukaemia(AML) cells to commonly used drugs and tool compounds. Methodologically it was envisioned to use CRISPR/Cas9 based genome editing to generate matched cell line pairs harbouring mutated or wild-type NPM1 genes whilst otherwise being identical. Further, automated high content microscopy would be used to study the differential effect of these drugs on the model cell lines. An integrated -omics approach would be used to understand differences in drug susceptibility in more detail and learn more about biology of NPM1. Due to unforeseen technical difficulties, the model system could not be established as planned. Rather an analogous research strategy was applied to study human immune processes. Whilst the original research aim could not be met, the training aims for the researcher could be achieved. The human immune system is a complex assembly of different cell types aimed at defending the host against invading pathogens. It is increasingly understood that the immune system plays an important role in controlling tumor growth. Tumors need to overcome the surveillance of the immune system to become macroscopically manifested and pharmacological interventions have been designed that can reactivate the immune system to treat cancer. When overactivated on the other hand, the human immune system can attack healthy cells of the body leading to autoimmune disease such as multiple sclerosis but also inflammatory diseases such as rheumatoid arthritis, ulcerative colitis etc. The societal cost of cancer and immune-related diseases combined is estimated to exceed EUR 200 Mio p.a. in the EU. The immune system is controlled in parts by soluble messenger factors (e.g. cytokines, chemokines) and by physical interactions of cells (“immunological synapses”,cell adherence processes etc.). So far the study of the immune system both in vitro and in animal models of disease have been limited to measuring soluble factors and events that are directly manifested in the expression of certain marker proteins on the surface of cells. The cell-cell interaction dimension until now has been very difficult to investigate. In the Superti-Furga lab a novel microscopy based approach has recently been developed that allows systematic measurement of cell-cell interactions in complex mixtures of immune cells ex vivo. This, for the first time, enables the study of the immune system directly in a cell-cell interaction dimension in high throughput in vitro. In a proof-of-concept study (Vladimer, Snijder et al, Nat.Chem.Biol.2017) it was shown that using the approach unexpected immunomodulatory properties of small molecule drugs could be identified. Specifically it was suggested that the TKI crizotinib could be combined with immunotherapy in a synergistic manner. For the purpose of the revised project the overall objective was in first place to better understand and systematically benchmark the novel cell-cell interaction readout by Vladimer, Snijder et al. We wanted to understand if certain stimuli elicited certain patterns of cell-cell interactions so that we could in reverse connect observed cell-cell interaction patterns of molecules of unknown function to known immune processes. In a second dimension, the objective was to understand if we could identify certain regulatory relationships based on our cell-cell interaction data. For example, if a certain gene is upregulated always together with another gene, the hypothesis can be set up that these genes are somehow functionally related. Similarly, we wanted to study if the occurrence of one cell-cell interaction could be functionally connected to the occurrence of another one. This could reveal important new insight into the wiring of the immune system.
During the course of the project the robustness of the method described by Vladimer, Snijder at al., was substantially improved. Two patent applications, one covering a novel type of plastic ware, one an improved analysis algorithm, were filed. Following, the effect of 37 different biological stimuli on the formation or reduction of cell-cell interactions was quantified in PBMCs of 6 different healthy donors. As hypothesized, we found that stimuli with similar effects gave rise to similar cell-cell interaction patterns. Similarly we found that certain cell-cell interactions always increased or decreased in a concerted manner. For example a cell type recently described as “killer dendritic cell”(“killer DC”) clustered together with conventional CD11c positive cells (likely myeloid DCs) when PBMCs were activated with certain stimuli such as IFNb or IL15. Work ongoing now is to functionally elucidate the cross talk between these cell types. For this purpose “killer DCs” and conventional myeloid DCs are being differentiated from monocytes ex vivo. Prior to maturation with appropriate stimulus (e.g. a TLR ligand) “killer DCs” and conventional myeloid DCs are separated and then stimulated either alone or in coculture. After that, cells are harvested, sorted, lysed and mRNA extracted. mRNA is then sequenced and compared between individually matured cells or cells matured in coculture. The fellow, after having moved from his post at CeMM to Allcyte GmbH, a startup company founded based on above described technology, is coordinating the continuation, conclusion and publication of the project by Dr. N. Meszaros, staff scientist, Superti-Furga lab. Overall aim is to position the publication as technology paper to demonstrate that analysis of coregulation patterns of cell-cell interactions can uncover new regulatory interactions between immune cells on a genetic and potentially also functional level.
The state of the art in the study of immunological processes in vitro has mostly been limited until now to i) measuring cytokine secretion and ii) measuring the expression of certain marker on the surface of cells or intracellularly (e.g. CD25 expression upon T-cell activation). We added a novel dimension of studying immune function by profiling cell-cell interactions and how they change in response to different stimuli in vitro. The project goes beyond state of the art by opening a completely new dimension of the study of immune processes in vitro. Further it brings concepts from gene network regulatory analysis to a cell-cell interaction level and the field of immunology. By the end of our investigations (to be concluded by Dr. Meszaros) we expect to have shown that i)specific immune stimuli give rise to specific cell-cell interaction patterns meaning that the measurement of cell-cell interaction patterns can be used to gain functional insight into immune processes and ii)co-regulatory patterns on cell-cell interaction level can be used to identify functional co-regulated processes between immune cells. Although the aforementioned “killer DCs” have been shown to have marked anti-tumor effects and have been upregulated in certain inflammatory diseases, it is at present not clear if we will be able to connect them functionally to any disease state. This may be done in future projects. The most tangible socio-economic impact of this project so far is the foundation of Allcyte GmbH, a company working on leveraging the above-mentioned technology to 1)develop biomarkers and in vitro diagnostic tests for personalized medicine applications and 2)support the development of new immunotherapy of cancer.
Coregulation networks