RAC2 Links B-Cell Receptor Signaling and Cell Adhesion in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an incurable non-Hodgkin B-cell lymphoma. B-cell receptor (BCR) signaling is chronically active in several mature B-cell malignancies including mantle cell lymphoma. Inhibition of the BCR and downstream pathway is highly effective in B-cell neoplasia and can be achieved at the cellular level through BTK inhibition by ibrutinib, for example. Ibrutinib has generated remarkable patients' response in large clinical trials and has been approved by FDA for the treatment of relapsed/refractory MCL. Ibrutinib targets Bruton tyrosine kinase primarily by inhibition of B-cell receptor signaling and cell proliferation, and secondarily by induction of tumor apoptosis. In addition, the drug inhibits the interaction of tumor cells with their microenvironment by disruption of cell adhesion. The effect on cell adhesion appears to be a mechanism shared by many BCR-targeted therapies including inhibitors of LYN (Leuk Res 38, 34, 2014), SYK (Blood, 115, 2578, 2010; Blood 125, 2336, 2015), and PI3K (idelalisib) (NEJM, 370, 997, 2014; Blood, 125, 2306). This class action raised a question regarding how the BCR pathway is connected to the cell adhesion phenotype. We set out to investigate the underlying mechanisms that are largely unknown at the present time.

Methods: We used RNA-sequencing to identify pathways that have differential responses to ibrutinib in MCL cell lines that are either intrinsically sensitive or resistant to ibrutinib. We then validate the RNA-Seq findings in sensitive and resistant cell lines via RNA interference and transfection, xenograft mouse models, and human primary MCL samples.

Results: We found that sensitivities of six MCL cell lines to ibrutinib correlated well with their cell adhesion phenotype. These include sensitive cell line Jeko-1, intermediate cell lines Mino, REC-1 and JVM2, and resistant lines Maver-1 and Granta 519. RNA-sequencing revealed that BCR signaling and cell adhesion transcriptional programs were coordinately down-regulated by ibrutinib in ibrutinib-sensitive cells, but not in ibrutinib-resistant cells. Among genes that are differentially regulated in the sensitive and resistant cell lines, we identified RAC2, a regulator of cell adhesion this is also involved in BCR signaling. To determine whether RAC2 is relevant, we examined RAC2 expression in primary MCL tumors by immunohistochemical staining (IHC). We found that RAC2 was weakly expressed in 7/10 classic MCL tumors and strongly expressed in 3/3 aggressive MCL. In contrast, in tonsilar lymphoid reactive tissues, RAC2 positivity was restricted only to mantle zone and T-zone of the follicles. For further validation, we found that RAC2 expression, at the protein level, was down-regulated by ibrutinib treatment in sensitive, but not resistant, cell lines. More interestingly, using co-immunoprecipitation, we found that RAC2 is physically associated with BLNK, an adaptor of early BCR signaling pathway, in the sensitive cells but not in the resistant cells. To establish that RAC2 is a regulator of MCL cell adhesion in the context of MCL, we demonstrated that siRNA knock-down of RAC2 impaired cell adhesion while exogenous introduction of RAC2 reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib showed slower tumor growth with reduced RAC2 expression by IHC. Lastly, we show that in primary human MCL cells, RAC2 was down-regulated by ibrutinib, and this down-regulation correlated well with cell adhesion impairment following ibrutinib treatment.

Conclusions: Our findings suggest the RAC2 mediates the link between BCR signaling and cell adhesion. Ibrutinib inhibits cell adhesion via down-regulation of RAC expression and via disruption of RAC2-BLNK interaction. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development.