The role of cannabinoid receptors, G alpha z, and B cell receptor in lymphoma pathobiology with focus on chemotaxis

Mantle cell lymphoma (MCL) and chronic lymphocytic leukaemia (CLL) are two incurable B cell malignancies, with an overall survival of 5 to 8 years and 6 to 10 years, respectively. Therapies are available but are often very aggressive, and patients relapse due to minimal residual disease. Minimal residual disease is defined by the presence of few malignant cells that escaped from therapy, mainly due to the survival signals provided by non-malignant cells from the tissue environment, in lymph nodes and in bone marrow. Alternative and targeted therapies are under investigation to increase patient overall survival and to reduce the risks of relapses. However, some patients do not respond to these treatments, as malignant cells develop mechanisms that prevent the drug efficacy. Many factors have already been depicted to contribute to MCL pathogenesis, and in this thesis, a new potential actor in MCL pathobiology is described, the protein G alpha z (Gαz).

The gene encoding for Gαz, GNAZ is overexpressed in most MCL cases compared to B lymphocytes from reactive lymph nodes. It was found that GNAZ expression correlates with lymphocytosis, and inversely correlates with the cannabinoid receptor type 1 previously described as a receptor potentially involved in the egress and/or retention of MCL cells within the tissue. Although the downregulation of GNAZ did not affect cell survival, proliferation or chemotaxis in vitro, its potential role in MCL pathobiology is of interest and needs further investigation.

Moreover, we characterize a co-culture in vitro system of MCL cell lines with mesenchymal stromal cells that permitted to identify differentially expressed genes between cells from different tissue origin. The JeKo-1 MCL cell line from peripheral blood origin, utilized the BCR signalling pathway to adhere to stromal cells, while the Rec-1 MCL cell line from lymph node origin did not, which conferred resistance to BCR targeted therapies. This system could be useful for testing patient samples to determinate a potential resistance before treatment decision.

Finally, the endocannabinoid system has been previously identified as dysregulated in both MCL and CLL. Here, we provide a new role of the endogenous cannabinoid 2-arachidonoylglycerol in chemotaxis of malignant B cells, regulated by both cannabinoid receptors type 1 and type 2. This endocannabinoid did not only induce chemotaxis by itself but also modulated the chemotaxis towards the chemokine CXCL12. In addition, a single administration of the natural cannabinoids, THC and CBD, in lymphoma patients promoted the redistribution of malignant cells from peripheral blood, and also affected non-malignant immune cells in blood. This potential negative effect of cannabinoids on the immune cells should be taken into consideration, knowing that around 25% of

List of scientific papers

I. Mundt F,* Merrien M,* Nygren L, Sutton LA, Christensson B, Wahlin BE, Rosenquist R, Sander B, Wasik AM. Expression of GNAZ, encoding the Gαz protein, predicts survival in mantle cell lymphoma. Br J Haematol. 2019 May;185(4):708-712. *First authors equal contribution.
https://doi.org/10.1111/bjh.15810

II. Sadeghi L,* Arvidsson G,* Merrien M, Wasik AM, Görgens A, Smith CIE, Sander B, Wright AP. Differential B-Cell Receptor Signaling Requirement for Adhesion of Mantle Cell Lymphoma Cells to Stromal Cells. Cancers (Basel). 2020 May 2;12(5):1143. *First authors equal contribution.
https://doi.org/10.3390/cancers12051143

III. Magali Merrien, Agata M. Wasik, Christopher M. Melén, Kristina Sonnevi, Henna-Riikka Junlén, Birger Christensson, Björn E. Wahlin and Birgitta Sander. 2-arachidonoylglycerol modulates the CXCL12-mediated chemotaxis in mantle cell lymphoma and chronic lymphocytic leukemia. [Manuscript]

IV. Christopher M. Melén*, Magali Merrien*, Agata M. Wasik, Georgios Panagiotidis, Olof Beck, Kristina Sonnevi, Henna-Riikka Junlén, Birger Christensson, Birgitta Sander**, Björn Engelbrekt Wahlin.** A single dose of cannabinoids transiently affects malignant B cells and CXCR4 expression in patients with indolent B-cell leukemia. *First authors equal contribution, **Last authors equal contribution. [Manuscript]