In vitro and in vivo evidence for uncoupling of BCR internalization and signaling in chronic lymphocytic leukemia

B-cell receptor activation, occurring within lymph nodes, plays a key role in the pathogenesis of chronic lymphocytic leukemia and is linked to prognosis. As well as activation of downstream signaling, receptor ligation triggers internalization, transit to acidified endosomes and degradation of ligand-receptor complexes. In the present study we investigated the relationship between these two processes in normal and leukemic B-cells. We found that leukemic B-cells, particularly anergic cases lacking the capacity to initiate downstream signaling, internalize and accumulate ligand in acidified endosomes more efficiently than normal B-cells. Furthermore, ligation of either surface CD79B, a Bcell receptor component required for downstream signaling, or surface IgM by cognate agonistic antibody, showed that the two molecules internalize independently of each other in leukemic but not normal B-cells. Since association with surface CD79B is required for surface retention of IgM, this suggests that uncoupling of B-cell receptor internalization from signaling may be due to dissociation of these two molecules in leukemic cells. Comparison of lymph node with peripheral blood cells from chronic lymphocytic leukemia patients showed that, despite recent B-cell receptor activation, lymph node B-cells expressed higher levels of surface IgM. This surprising finding suggests that the B-cell receptors of lymph node and peripheral blood derived leukemic cells might be functionally distinct. Finally, long-term therapy with the Bruton’s tyrosine kinase inhibitors ibrutinib or acalabrutinib resulted in a switch to an anergic pattern of B-cell receptor function with reduced signaling capacity, surface IgM expression and more efficient internalization. Introduction It is now clear that signaling through the BCR plays a key role in the pathogenesis of CLL and other lymphomas. Several components of this pathway including: Syk,(1) Erk,(2) Akt,(3) NFAT(4) and NFκB(5) can be constitutively activated and drugs that target BCR signaling, such as the Bruton’s tyrosine kinase inhibitors (BTKi), ibrutinib and acalabrutinib, are proving extremely effective in the clinic. (6, 7) BCR responsiveness varies markedly between patients with CLL and is linked to prognosis. (8) Some cases show features of anergy,(4, 9) a pattern that is associated with lack of ability to transduce a downstream signal in response to BCR ligation and the presence of markers of good prognosis, including low levels of CD38 and mutated immunoglobulin heavy-chain variable (IGHV) genes. In contrast, cases with responsive or signaling competent BCRs usually express high levels of CD38, have unmutated IGHV genes and a more unfavorable clinical course;(10) interestingly these patients tend to respond more rapidly to BCR antagonists than those with anergic BCRs. Although BTKi therapy is very successful in controlling CLL, it is not curative and many patients are left with low level residual disease, which regrows on discontinuation of drug or when resistance mutations develop. (11, 12) This persistent disease also suggests that, within individual patients, the tumor may not behave in a homogeneous manner. (13) Despite the central importance of BCR signaling in CLL and the efficacy of drugs that block this pathway, there is relatively little known about BCR dynamics in leukemic B-cells. Surface levels of IgM and other BCR components are generally lower in CLL compared to normal B-cells and it has been suggested that this might be due to a failure to properly assemble the sIg α/β subunits CD79A and CD79B. (14) Recent studies have shown that total IgM and CD79A levels are near normal in CLL but that CD79B expression, which is required for transport of BCR to the cell surface,(15) is reduced thus trapping IgM within the cell. (16) Exposure to IL4 increases CD79B expression and allows sIgM levels to increase and BCR signaling capacity to improve. (16, 17) CLL cell surface BCRs have an immature pattern of glycosylation that matures following ex-vivo incubation(18) or exposure to IL4(17) in keeping with accelerated BCR turnover induced by chronic activation. It has also been reported that, within the PB of individual patients with CLL, leukemic cells with the lowest sIgM expression show biochemical features of recent activation and proliferation, presumably because they have recently been released from lymphoid tissues where BCR stimulation and activation are thought to occur. (19, 20) Taken together, these previous data suggest that the reduced sIgM levels observed in CLL are due to a combination of increased turnover consequent on chronic activation coupled with defective transport to the cell surface resulting from a deficiency of CD79B. The ability of CLL BCRs to become internalized also has implications for how the tumor interacts with other cells, such as T-cells. We, and others, have previously shown that, as in normal LNs, activated CD4+ T-cells co-localize with proliferating tumor cells and, in-vitro, can supply signals that cause tumor proliferation. (21-24) This process normally involves endocytosis and processing of antigen bound to BCR, however it is not known whether CLL B-cells are capable of providing this function. In the present study we investigated whether, like normal B-cells, CLL cells can internalize their BCR and to what extent this is linked to downstream signaling in both untreated patients and those receiving therapy with the Bruton’s Tyrosine Kinase inhibitors ibrutinib and acalabrutinib. Our results shed new light on BCR function in CLL and have implications for understanding the mode of action of this important new class of drugs.