Center for Molecular Medicine Cologne

Michael Hallek / Phoung-Hien Nguyen - A 3

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

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 the non-receptor tyrosine kinase Lyn in the tumor microenvironment promoting CLL.

Introduction

Chronic lymphocytic leukemia (CLL) represents the most common B cell malignancy in the aging populations that is characterized by the accumulation of mature CD5-positive B cells in the blood and lymphoid tissues of affected patients (Hallek et al., Lancet 2018). Binding of antigens to the B cell receptor (BCR) facilitates in the formation and signal initiation of the BCR signalosome consisting of LYN, SYK, BTK and other kinases, which are recognized as key factors in both CLL pathogenesis and progression. 

The last decade has witnessed tremendous improve-ment in CLL therapy with the approval of various novel inhibitors targeting the BCR signaling pathway. The approval of BCR inhibitors such as ibrutinib (BTK inhibitor) and idelalisib (PI3K-δ) has been changing the field of CLL therapy significantly (Hallek et al., Blood 2018). Particularly, treatment options for CLL has recently moved towards chemo-free regiment with the combination of targeted therapy and/or immunotherapy (Jain et al., NEJM 2019; Fischer et al., NEJM 2019).

1.  New perspectives for alternative targeting of tyrosine kinases in accessory cells besides directly targeting malignant cells.

A hallmark of CLL is the strong dependency of the malignant B cells on the tumor microenvironment (TME), which provides CLL cells with pivotal survival support (Nguyen et al., Leukemia 2019). Using the most validated animal model for CLL, our research group has contributed to the understanding of CLL cell survival and how different factors of the TME affect CLL progression in vivo(Nguyen et al., Cancer Cell 2016; Fedorchenko et al., Blood 2013; Reinart et al., Blood 2013).

Strikingly, clinical observations have provided a paradigm shift in our understanding of the role of BCR kinases in B cell malignancies. The class effect of BCR inhibitors is not characterized by their tumor 

killing capacity, but rather by a slow redistribution of malignant cells from the protective niche to the peri-phery. In addition, the use of genetically engineered murine model systems revealed the surprising actions of these kinases in the formation of a TME nurturing CLL cells. These evidence generated a change of perspective in that targeting these kinases in accessory cells rather than leukemic cells is necessary to create effective therapies (Figure 1).

2.  Survival of malignant B cells relies on the microenvironmental expression of Lyn kinase.

Lyn kinase belongs to the Src family of non-receptor tyrosine kinases, and was initially identified as a crucial BCR modulator, regulating both positive and negative signaling pathways following BCR activation (Hibbs et al., Cell 1995). 

We investigated the role of Lyn on the in vivo level by analyzing leukemic development in a Lyn-knockout CLL mouse model. 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 was due to the loss of Lyn kinase in the micro-environmental cells. We could show that the Lyn-knockout 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 (Figure 2). 

Moreover, we could observe similar effects of the Lyn-associated TME in a highly aggressive Burkitt’s lymphoma like model. Lyn-knockout recipients developed delayed lymphoma burden, reduced lymphadenopathy and splenomegaly, and prolonged survival after transplantation with these lymphoma cells.

Our results identified for the first time an unexpected functional role of Lyn in the TME of B cell lymphoma, opening new therapeutic avenues by targeting the TME in these malignancies.

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

1.   Fischer K, Al-Sawaf O, Bahlo J, Fink AM, et al., and Hallek M: Venetoclax and Obinutuzumab in Patients with CLL and Coexisting Conditions (2019). N Engl J Med 380: 2225-2236

2.   Nguyen PH*, Niesen E*, and Hallek M: New roles for B cell receptor associated kinases: when the B cell is not the target (2019). Leukemia 33(3):576-587

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

4.   Hallek M,Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, et al.: iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL (2018). Blood 131:2745-2760. 

5.   Nguyen PH, Fedorchenko O, Rosen N, Koch M, et al., and Hallek M. LYN Kinase in the Tumor Microenvironment Is Essential for the Progression of Chronic Lymphocytic Leukemia(2016). Cancer Cell 30: 610-622

Bachurski, D., Schuldner, M., Nguyen, P.H., Malz, A., Reiners, K.S., Grenzi, P.C., Babatz, F., Schauss, A.C., Hansen, H.P., Hallek, M., and Pogge von Strandmann, E. (2019). Extracellular vesicle measurements with nanoparticle tracking analysis - An accuracy and repeatability comparison between NanoSight NS300 and ZetaView. J Extracell Vesicles 8, 1596016.

Chmielewski, M., Kuehle, J., Chrobok, D., Riet, N., Hallek, M., and Abken, H. (2019). FimH-based display of functional eukaryotic proteins on bacteria surfaces. Sci Rep 9, 8410.

Drossler, L., Lehmann, C., Topelt, K., Nierhoff, D., Vehreschild, J.J., Rybniker, J., Hallek, M., Fischer, J., Stormberg, V., Fatkenheuer, G., Wieland, U., and Jung, N. (2019). HBsAg-negative/anti-HBc-positive patients treated with rituximab: prophylaxis or monitoring to prevent hepatitis B reactivation? Infection 47, 293-300.

Fischer, K., Al-Sawaf, O., Bahlo, J., Fink, A.M., Tandon, M., Dixon, M., Robrecht, S., Warburton, S., Humphrey, K., Samoylova, O., Liberati, A.M., Pinilla-Ibarz, J., Opat, S., Sivcheva, L., Le Du, K., Fogliatto, L.M., Niemann, C.U., Weinkove, R., Robinson, S., Kipps, T.J., Boettcher, S., Tausch, E., Humerickhouse, R., Eichhorst, B., Wendtner, C.M., Langerak, A.W., Kreuzer, K.A., Ritgen, M., Goede, V., Stilgenbauer, S., Mobasher, M., and Hallek, M. (2019). Venetoclax and Obinutuzumab in Patients with CLL and Coexisting Conditions. N Engl J Med 380, 2225-36.

Nguyen, P.H., Niesen, E., and Hallek, M. (2019). New roles for B cell receptor associated kinases: when the B cell is not the target. Leukemia 33, 576-87.

Pflug, N., Cramer, P., Robrecht, S., Bahlo, J., Westermann, A., Fink, A.M., Schrader, A., Mayer, P., Oberbeck, S., Seiler, T., Zenz, T., Durig, J., Kreuzer, K.A., Stilgenbauer, S., Eichhorst, B., Hallek, M., Herling, M., and Hopfinger, G. (2019). New lessons learned in T-PLL: results from a prospective phase-II trial with fludarabine-mitoxantrone-cyclophosphamide-alemtuzumab induction followed by alemtuzumab maintenance. Leuk Lymphoma 60, 649-57.

Rossi, A., Dupaty, L., Aillot, L., Zhang, L., Gallien, C., Hallek, M., Odenthal, M., Adriouch, S., Salvetti, A., and Buning, H. (2019). Vector uncoating limits adeno-associated viral vector-mediated transduction of human dendritic cells and vector immunogenicity. Sci Rep 9, 3631.

Golfmann K, Meder L, Koker M, Volz C, Borchmann S, Tharun L, Dietlein F, Malchers F, Florin A, Buttner R, Rosen N, Rodrik-Outmezguine V, Hallek M, and Ullrich RT (2018). Synergistic anti-angiogenic treatment effects by dual FGFR1 and VEGFR1 inhibition in FGFR1-amplified breast cancer. Oncogene 37, 5682-5693.

Hahn M, Burckert JP, Luttenberger CA, Klebow S, Hess M, Al-Maarri M, Vogt M, Reissig S, Hallek M, Wienecke-Baldacchino A, Buch T, Muller CP, Pallasch CP, Wunderlich FT, Waisman A, and Hovelmeyer N (2018). Aberrant splicing of the tumor suppressor CYLD promotes the development of chronic lymphocytic leukemia via sustained NF-kappaB signaling. Leukemia 32, 72-82.

Herling CD, Abedpour N, Weiss J, Schmitt A, Jachimowicz RD, Merkel O, Cartolano M, Oberbeck S, Mayer P, Berg V, Thomalla D, Kutsch N, Stiefelhagen M, Cramer P, Wendtner CM, Persigehl T, Saleh A, Altmuller J, Nurnberg P, Pallasch C, Achter V, Lang U, Eichhorst B, Castiglione R, Schafer SC, Buttner R, Kreuzer KA, Reinhardt HC, Hallek M, Frenzel LP, and Peifer M (2018). Clonal dynamics towards the development of venetoclax resistance in chronic lymphocytic leukemia. Nat Commun 9, 727.

Meder L, Schuldt P, Thelen M, Schmitt A, Dietlein F, Klein S, Borchmann S, Wennhold K, Vlasic I, Oberbeck S, Riedel R, Florin A, Golfmann K, Schlosser HA, Odenthal M, Buttner R, Wolf J, Hallek M, Herling M, von Bergwelt-Baildon M, Reinhardt HC, and Ullrich RT (2018). Combined VEGF and PD-L1 blockade displays synergistic treatment effects in an autochthonous mouse model of small cell lung cancer. Cancer Res10.1158/0008-5472.CAN-17-2176.

Pflug N, Cramer P, Robrecht S, Bahlo J, Westermann A, Fink AM, Schrader A, Mayer P, Oberbeck S, Seiler T, Zenz T, Durig J, Kreuzer KA, Stilgenbauer S, Eichhorst B, Hallek M, Herling M, and Hopfinger G (2018). New lessons learned in T-PLL: results from a prospective phase-II trial with fludarabine-mitoxantrone-cyclophosphamide-alemtuzumab induction followed by alemtuzumab maintenance. Leuk Lymphoma10.1080/10428194.2018.1488253, 1-9.

Schrader A, Crispatzu G, Oberbeck S, Mayer P, Putzer S, von Jan J, Vasyutina E, Warner K, Weit N, Pflug N, Braun T, Andersson EI, Yadav B, Riabinska A, Maurer B, Ventura Ferreira MS, Beier F, Altmuller J, Lanasa M, Herling CD, Haferlach T, Stilgenbauer S, Hopfinger G, Peifer M, Brummendorf TH, Nurnberg P, Elenitoba-Johnson KSJ, Zha S, Hallek M, Moriggl R, Reinhardt HC, Stern MH, Mustjoki S, Newrzela S, Frommolt P, and Herling M (2018). Actionable perturbations of damage responses by TCL1/ATM and epigenetic lesions form the basis of T-PLL. Nat Commun 9, 697.

Hahn M, Burckert JP, Luttenberger CA, Klebow S, Hess M, Al-Maarri M, Vogt M, Reissig S, Hallek M, Wienecke-Baldacchino A, Buch T, Muller CP, Pallasch CP, Wunderlich FT, Waisman A, and Hovelmeyer N (2017). Aberrant splicing of the tumor suppressor CYLD promotes the development of chronic lymphocytic leukemia via sustained NF-kappaB signaling. Leukemia 10.1038/leu.2017.168.

Knittel G, Rehkamper T, Korovkina D, Liedgens P, Fritz C, Torgovnick A, Al-Baldawi Y, Al-Maarri M, Cun Y, Fedorchenko O, Riabinska A, Beleggia F, Nguyen PH, Wunderlich FT, Ortmann M, Montesinos-Rongen M, Tausch E, Stilgenbauer S, L PF, Herling M, Herling C, Bahlo J, Hallek M, Peifer M, Buettner R, Persigehl T, and Reinhardt HC (2017). Two mouse models reveal an actionable PARP1 dependence in aggressive chronic lymphocytic leukemia. Nat Commun 8, 153.

Schiffmann LM, Brunold M, Liwschitz M, Goede V, Loges S, Wroblewski M, Quaas A, Alakus H, Stippel D, Bruns CJ, Hallek M, Kashkar H, Hacker UT, and Coutelle O (2017). A combination of low-dose bevacizumab and imatinib enhances vascular normalisation without inducing extracellular matrix deposition. Br J Cancer 116, 600-8.

Shimabukuro-Vornhagen A, Garcia-Marquez M, Fischer RN, Iltgen-Breburda J, Fiedler A, Wennhold K, Rappl G, Abken H, Lehmann C, Herling M, Wolf D, Fatkenheuer G, Rubbert-Roth A, Hallek M, Theurich S, and von Bergwelt-Baildon M (2017). Antigen-presenting human B cells are expanded in inflammatory conditions. J Leukoc Biol 101, 577-87.

Wennhold K, Thelen M, Schlosser HA, Haustein N, Reuter S, Garcia-Marquez M, Lechner A, Kobold S, Rataj F, Utermohlen O, Chakupurakal G, Theurich S, Hallek M, Abken H, Shimabukuro-Vornhagen A, and von Bergwelt-Baildon M (2017). Using Antigen-Specific B Cells to Combine Antibody and T Cell-Based Cancer Immunotherapy. Cancer Immunol Res 5, 730-43.

Prof. Dr. Michael Hallek CMMC Cologne
Prof. Dr. Michael Hallek

Dept. I of Internal Medicine / RG location - CECAD Building

Principal Investigator A 3

Executive Board Member

+49 221 478 4400

+49 221 478 5455

Dept. I of Internal Medicine / RG location - CECAD Building

Kerpener Str. 62

50937 Cologne

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

CMMC Profile Page

Curriculum Vitae (CV)

Publications - Michael Hallek

Link to PubMed

Dr. Hien Nguyen CMMC Cologne
Dr. Hien Nguyen

Dept. I for Internal Medicine / RG location - CECAD Building

Co-Principal Investigator A 3

+49 221 478 84120

+49 221 478 32400

Dept. I for Internal Medicine / RG location - CECAD Building

Joseph-Stelzmann Str. 26

50931 Cologne

CMMC Profile Page

Publications - Hien Nguyen

Link to PubMed

Group Members

Michael Hallek (Group Leader)
Oleg Fedorchenko (PostDoc)
Phuong-Hien Nguyen (PostDoc)
Hinrich Hansen (PostDoc)
Rocio Rebollido-Rios (PostDoc)
Maximilian Koch (PhD student)
Sebastian Reinartz (PhD student)
Viktoria Kohlhas (PhD student)
Daniel Bachurski (MD-PhD student)
Thais Dolzany De Oliveira (PhD student)
Maria José Fariña-Morillas (PhD student)
Hedwig Göx (MD student)
Alexander F. vom Stein (MD student)
Lea Reemann (MD student)
Natascha Rosen (Technician)
H. Bohner (Technician)
Carina Folger (Technician)

Figure 1
Figure 2