1038 - Inhibition of G protein-coupled receptor kinase 2 (GRK2) as a strategy to modulate leukemic cell homing and activation in CLL

Proliferation of leukemic cells and resistance to therapy occur within lymphoid tissues, supporting the idea that the retention of leukemic cells within this tumour microenvironment contributes to disease progression and relapse. GRK2 plays a central role in B cell homing to lymphoid organs by inducing Sphingosine-1 phosphate receptor-1 (S1PR1) downregulation, which allows lymphocytes to overcome the S1P-mediated retention in the blood and to enter into lymphoid tissues. Migration towards the high concentration of S1P in the vascular compartment also mediates B cell exit from lymphoid tissues. In mouse models, GRK2-deletion on B cells leads to an increased response to S1P and thus a higher presence of B cells in peripheral blood over bone marrow, lymph nodes and spleen follicles (Arnon TI, et al. 2011. Science). In addition, GRK2 has been implicated in signalling pathways related to cancer progression (Nogués L, et al. 2017. Molecular Pharmacology). The expression and function of GRK2 in CLL have not been studied yet. Here, we aim to evaluate the role of GRK2 in leukemic cell migration, activation and survival.

Primary leukemic B cells were obtained from peripheral blood of untreated CLL patients and normal B cells from healthy volunteers. GRK2 expression was evaluated by western blot and qRT-PCR. Cell activation and drug-induced apoptosis were evaluated by flow cytometry. Chemotaxis towards S1P, CXCL12 or CXCL13 was evaluated in vitro using a Transwell cell migration assay. Murine leukemic B cells were obtained from Eµ-TCL-1 mice (C57BL/6 background). GRK2-deficient murine leukemic cells were generated by CRISPR/Cas9 editing on the mouse cell line TCL1-355 TKO, as previously described (Chakraborty S, et al. 2021. Blood). For in vivo migration assays, control and GRK2-deficient murine leukemic cells were labeled with CFSE or CTV, mixed in a ratio 1:1 and injected through the tail vein. Twenty hours later, their localization in peripheral blood, bone marrow and spleen was evaluated by flow cytometry. GraphPad Prism software was used for statistical analysis.

We found that leukemic cells from CLL patients express similar levels of GRK2 as B cells from healthy volunteers (Figure 1A-C). In vitro treatment of leukemic cells with the GRK2 inhibitor, CMPD101, did not affect spontaneous or venetoclax-induced apoptosis (not shown). The presence of CMPD101 did not counteract the up-regulation of CD69 expression on CLL cells activated through the BCR (Figure 1D). On the other hand, when CLL cells were activated by co-culturing with autologous activated T cells, the presence of CMPD101 decreased the up-regulation of the activation marker CD86 on leukemic B cells, which was accompanied with decreased CD40L expression on T cells and a lower capacity to secrete IL-10 (Figure E-H). We then evaluated the effect of GRK2 inhibition on the in vitro migratory response of CLL cells towards S1P, CXCL12 and CXCL13. We found that the response of CLL cells to S1P was significantly increased by CMPD101 (Figure 1I), while migration towards chemokines was not affected (Figure 1J). CMPD101 also increased migration towards S1P of leukemic cells obtained from peripheral blood or lymphoid tissues of Eµ-TCL1 mice (Figure 1K). Moreover, using CRISPR/Cas9 technology on the TCL1-355 TKO cell line, we generated GRK2-deficient murine leukemic cells (GRK2KO) (Figure 2A) and their control, MOCK-transfected, leukemic cells (MOCK), which allowed us to show that GRK2 deletion increased the in vitro response of leukemic cells to S1P (Figure 2B). As expected, CMPD101 increased S1P response of MOCK but not on GRK2KO cells (not shown). Migration towards CXCL12 and CXCL13 was similar between GRK2KO and MOCK cells (not shown). Interestingly, GRK2KO cells showed a slightly lower spontaneous proliferation rate when cultured in vitro compared to MOCK cells, which was evident after day 8 of culture (Figure 2C), and also proliferated less in response to LPS (Figure 2D). Finally, we evaluated the in vivo homing capacity of GRK2KO and MOCK cells intravenously injected into mice. At 20 hs post-injection, we found that GRK2KO cells preferentially localized in blood and spleen and were underrepresented in the bone marrow, compared to MOCK cells (Figure 2E).

Our results suggest that GRK2 inhibition could be explored as a strategy to induce their retention in the blood, increasing their exposure to therapeutic agents and/or to overcome resistance to treatment induced by the protective microenvironment.