Proliferative Effect of APRIL On Primary Multiple Myeloma Cells Is Dictated by D-Type Cyclin Group and Translocation Status.

Understanding how multiple myeloma (MM) cells proliferate and self-renew is crucial to treating resistant disease and preventing relapse. The TNF family members APRIL (A proliferation-inducing ligand) and BAFF (B-cell activating factor) are secreted in the MM bone marrow microenvironment and share two common receptors, TACI and BCMA. APRIL and BAFF and have been shown to exert a mainly anti-apoptotic effect in human myeloma cell lines (HMCLs). Little is known, however, about the proliferative effect of APRIL and BAFF on primary MM cells. Aims: We aimed to determine the effect of APRIL and BAFF on cell cycle progression in primary MM cells and on the modulation of D-type cyclins and other cell cycle proteins. Patients and Methods: We studied the effects of APRIL and BAFF on purified MM cells from 26 patients by flow-cytometry using surface CD138 / Ki67 / propidium iodide staining. A culture system was optimised in which a mean (±SEM) of 73.3±10.9% (n=23) of primary CD138+ MM cells survived for up to 3 days in vitro. D-type cyclin expression was assessed by immuno-histochemistry (IHC) ± western blotting and correlated with FISH for IgH translocations. Fourteen patients expressed cyclin D1, 4 in association with t(11;14); while 12 patients expressed cyclin D2, 1 with t(4;14) and 4 with t(14;16). TACI and BCMA expression were determined by flow cytometry. Western blotting was used to determine expression and modulation of cell cycle proteins and activation of signalling pathways in vitro. Membrane-bound APRIL was detected by IHC in MM trephine biopsies. Results: In-vitro culture with APRIL for 72 hours increased the overall percentage of CD138+ cells in S+G2/M phases (S/G2M) from a mean (±SEM) of 5.5 ± 1.1% to 8.1 ± 1.5% (p<0.05). However, cyclin D2 expressing CD138+ cells demonstrated a greater response to APRIL than D1 expressing cells. In the D2 group (n=12), APRIL increased the S/G2M fraction from 8.3 ± 2.1% to 13.5 ± 2.2%, (p<0.01), while in the D1 group (n=14), proliferation was unaffected (3.0 ± 0.5%, compared with 3.1 ± 0.7% in control). Similar results were obtained when absolute numbers of CD138+ cells in S/G2M were analysed. Proliferative response to APRIL was more marked in those cases with IgH translocations (S/G2M fraction increased from 6.4±1.6% to 14.8±2.0%, p<0.001). In comparison with APRIL, BAFF had a lesser effect, increasing the S/G2M fraction in the D2 group from 8.3 ± 2.1% in control to 11.7 ± 3.5%, p=0.05). BAFF had no significant effect on proliferation in the D1 group. Results were verified by increased ³H-thymidine and bromodeoxyuridine uptake. APRIL-induced proliferation was maximal after 48-72 hours of incubation, was dose-dependent (maximal effect at 200ng/ml) and completely inhibited by TACI-Fc. In the D2 group, proliferation in response to APRIL was accompanied by increased expression of cyclin D2, cdk4, cdk6 and phospho-pRB by western blotting. In contrast, in the D1 group, neither cyclin D1 nor other cell cycle regulators was affected by APRIL. Proliferative response to APRIL was accompanied by increased expression of phospo-PKB. APRIL-induced proliferation showed a positive correlation with TACI expression (p<0.01), but not with BCMA. In contrast to the cell cycle effect, APRIL had no significant effect on survival of primary CD138+ cells either in D2 or D1 groups (viable cell number was 91±22%, and 129±18% of control respectively). IHC revealed the presence of membrane-bound APRIL on MM cells in BM sections from both D1 and D2 patients. IHC was used to examine expression of cell cycle regulators in vivo. In 5 cases where cyclin D1 expression was associated with proliferation, ie phospho-pRb expression, strong expression of cdk6 was seen, with weaker expression of cdk4. Expression of cyclin D2 was always accompanied by phospho-pRb expression (n=5), with strong expression of both cdk4 and -6 in 3, and just cdk6 in 2. Conclusions: APRIL stimulates the proliferation of cyclin D2 expressing primary MM cells, in particular those associated with known IgH translocations, but has minimal effect on cells expressing cyclin D1. These findings suggest that MM cells from different cyclin D/translocation classes rely on different stimuli for proliferation and cell-cycle re-entry.

No relevant conflicts of interest to declare.