Michael Hallek / Phoung-Hien Nguyen - A 3

Comprehensive analyses of the mechanistic role of LYN kinase in B cell malignancies

Summary

Chronic lymphocytic leukemia (CLL) is a malignant disease in which the survival of leukemic cells is strongly dependent of continuous stimulation by extracellular factors. We aim to explore this potential "Achilles heel" of CLL using a CLL mouse model and we work to develop novel therapeutic approaches in the future. By using gene knockout mouse models, we analyze the role of non-receptor tyrosine kinases, of surface receptors and of cytokines/chemokines in the development and promotion of CLL.

Introduction

Chronic lymphocytic leukemia (CLL) is the most frequently occurring leukemia that is characterized by the accumulation of mature CD5-positive B cells in the blood and lymphoid tissues of affected patients. Despite improvements in CLL treatment, this disease remains incurable. Novel inhibitors of kinases (Idelalisib, Ibrutinib) or of BCL-2 (Venetoclax) have further improved the options of CLL therapy, but their definitive curative potential remains to be determined by clinical trials testing combinations. Various cellular components from the surrounding niche provide CLL cells with a permissive environment to survive (Fig.1). These “hosting” components and the CLL cells communicate and mutually influence each other’s features and activities to ensure CLL manifestation.

Lyn kinase in the tumor microenvironment is essential for the progression of CLL

The recent advent of inhibitors that are supposed to target BCR signaling has substantially improved therapeutic options for CLL. Although targeting kinases of the BCR complex with novel inhibitors has provided potent drugs for CLL patients such as Ibrutinib or Idelalisib, the mode of action of these inhibitors is far from being fully understood. Moreover, recent sequencing of whole exome in a large cohort of CLL detected no mutation in the BCR associated kinases such as LYN, SYK or BTK, suggesting that these kinases are not the dominant drivers of CLL cell transformation. Thus we investigated the role of LYN on the in vivo level by analyzing CLL development in Eµ-TCL1;Lyn-/- mice. Complete deletion of LYN resulted in significantly reduced CLL cells in the peripheral blood and decreased CLL infiltration into the lymphoid tissues of mice. Surprisingly, the reduced burden of CLL in Eµ-TCL1;Lyn-/- mice was due to the loss of LYN in the microenvironmental cells. We could show that the Lyn-/- mice did not support the engraftment of murine CLL cells in a syngeneic transplantation model, leading to a delayed CLL progression and prolonged survival compared to wild type recipients. Our results indicate a striking function of LYN and of its substrate BTK in microenvironmental cells that is essential for CLL manifestation in vivo. These results also generate a change of perspective in that tyrosine kinases such as LYN or BTK might need to be targeted in accessory cells such as macrophages rather than in leukemic B cells to create effective therapies (Figure 1). 

We could observe similar effects of the Lyn-associated TME in another model besides the Eµ-TCL1 model. Here, we used the Myc-induced Burkitt’s lymphoma like model and transplant lymphoma cells into wildtype (WT) and Lyn-/- littermates. Lyn-/- recipients developed delayed lymphoma burden, reduced lymphadenopathy and splenomegaly (Figure 2), and prolonged survival. Our results identified for the first time an unexpected functional role of Lyn in the TME of B cell malignancies, opening new therapeutic avenues by targeting the TME in these malignancies.

Establishment of a fully human coculture model to study the role of macrophages in CLL pathogenesis.

Given the pivotal role of macrophages in CLL pathogenesis, we recently established a novel coculture system of CLL with the differentiated THP 1 macrophage cell lines, which significantly support CLL cell viability in vitro (Figure 3). Transcriptomic and proteomic analyses were applied to provide insights into the Lyn-dependent downstream signaling pathways in the feeder macrophages. Our results suggest that Lyn-knockout in these cells induces unexpected widespread molecular changes, highlighting the necessity to further dissect the involved pathways mechanistically. We are now further characterizing the role of LYN in these LYN negative cell lines to study their interaction with leukemic cells regarding their nursing, adhesion and migration capacity.

Perspectives 

Our project aims to provide more insights into the functional role of non-receptor tyrosine kinase LYN in the microenvironmental cells, and in the malignant B cells. Our specific goals will foster a more complete understanding of the role of this kinase, as well as the mechanistic action of kinase inhibitors, formerly believed to target the leukemic cells. The overall goal is to facilitate new development of treatment combinations to create effective therapies for B cell malignancies

Selected publications

Nguyen PH, Fedorchenko O, Rosen N, Koch M, Barthel R, Winarski T, Florin A, Wunderlich FT, Reinart N, and Hallek M (2016). LYN Kinase in the Tumor Microenvironment Is Essential for the Progression of Chronic Lymphocytic Leukemia. Cancer Cell 30, 610-622. 

Hallek M, Shanafelt TD, Eichhorst B (2018). Chronic lymphocytic leukaemia. Lancet 14;391(10129):1524-1537. 

Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, Hillmen P, Keating M, Montserrat E, Chiorazzi N, Stilgenbauer S, Rai KR, Byrd JC, Eichhorst B, O'Brien S, Robak T, Seymour JF, Kipps TJ (2018). Guidelines for diagnosis, indications for treatment, response assessment and supportive management of chronic lymphocytic leukemia. Blood. [Epub ahead of print].


Prof. Dr. Michael Hallek

Dept. I of Internal Medicine

Prof. Dr. Michael Hallek

Principal Investigator A 3
Executive Board Member

michael.hallek@uni-koeln.de

Work +49 221 478 4400

Fax (Work) +49 221 478 5455

Dept. I of Internal Medicine
Kerpener Str. 62
50937 Cologne

http://innere1.uk-koeln.de/team

Publications - Michael Hallek

Link to PubMed


Dr. Hien Nguyen

Dept. I for Internal Medicine

Dr. Hien Nguyen

Co-Principal Investigator A 3

hien.nguyen@uk-koeln.de

Work +49 221 478 84120

CECAD Research Center
Joseph-Stelzmann Str. 26
50931 Cologne

Publications - Hien Nguyen

Link to PubMed

Group Members

Michael Hallek (Group Leader)
Phuong-Hien Nguyen (PostDoc)
Oleg Fedorchenko (PostDoc)
Maximilian Koch (Doctoral student)
Sebastian Reinartz (Doctoral student)
Viktoria Kohlhas (Doctoral student)
Julia Göx (MD student)
Alexander F. vom Stein (MD student)
Natascha Rosen (Technician)
H. Bohner (Technician)
Carina Folger (Technician)

Figure 1
Lyn is essential for the formation of a leukemia supporting niche (Nguyen et al., Cancer Cell 2016)
Figure 2
Delayed progression of B cell lymphoma in Lyn-knockout recipients, showing in the reduced volume of pathologic lymph nodes (upper panel) and of the spleen (lower panel) after tumor transplantation.
Figure 3
THP-1 macrophages potently support the survival of primary CLL cells in coculture.