Abstract

Maintenance of the G0 state is a key to survival of CLL B-cells. Heterogeneity of prognosis suggests that CLL is not a uniform disease. Molecules expressed in CLL with unfavorable prognosis, such as ZAP-70, Lyn, CD38 and others, provide stimuli which, coupled with B-cell receptor signaling, may alter cell cycle progression and delay apoptosis. We studied mechanisms of apoptosis in CLL B-cells via inhibition of Dipeptidyl Peptidase 2 (DPP2). DPP2 is a serine protease cloned in our lab, which is involved in the maintenance of the G0 (quiescent) state. Inhibition of DPP2 triggers apoptosis in healthy B-cells. 50 patients with B-CLL were recruited from the Hematology clinics at Tufts-NEMC (Boston, MA). Median time from diagnosis of CLL to enrollment in the study was 7 years with median follow up of 10.5 years. 24 patients (48%) have received treatment in the course of their disease. CLL B-cells were isolated from peripheral blood with standard Ficoll-Hypaque technique, treated with ValboroPro (Point Therapeutics), a non-specific inhibitor of DPPs, and/or AX8819 (ActivX), a DPP2-specific inhibitor, incubated for 16 hours and stained with anti-CD19 antibodies, propidium iodide and Annexin V. Expression of DPP2 and ZAP-70 mRNA was assessed by RT-qPCR, p27 protein - by western blot analysis. By blocking DPP2 protease activity, we distinguished two subsets of CLL - sensitive (S-CLL) and resistant (R-CLL) to DP-P2 inhibition-induced apoptosis. In 30 cases (60%), inhibition of DPP2 resulted in caspase-dependent apoptosis of CLL B-cells (S-CLL). 70–90% of B-cells stained Annexin V-positive after incubation with AX8819. Pre-incubation with Rituximab (Biogen Idec) did not enhance cell death. In the remaining 20 cases (40%) inhibition of DPP2 did not cause cell death (R-CLL). R-CLL demonstrated higher expression of ZAP-70 mRNA (p<0.001) and lower levels of p27 protein (p<0.01) than S-CLL. ZAP-70 mRNA levels strongly correlated with resistance to apoptosis (χ2=26.7, p<0.0001). Inhibition of DPP2 resulted in a decrease in p27 protein levels in S-CLL, but not in R-CLL. Both groups expressed higher DPP2 mRNA levels than B-cells derived from healthy donors. In the R-CLL subgroup, 18 patients (84%) required treatment for their disease, 14 received more than one treatment regimen, 2 underwent allogeneic transplant and 2 died of complications of CLL. Among the S-CLL cohort, 10 patients (33%) initiated treatment, 4 received more than one treatment regimen. R-CLL cohort required treatment earlier than S-CLL (3.5 vs. 9.9 years from diagnosis). Thus, DPP2 inhibition discriminates two subsets of CLL based on their ability to undergo apoptosis upon disruption of the quiescent program. We postulate that DPP2 is critical for the maintenance of G0 in S-CLL. Its inhibition leads to inappropriate cell cycle entry, as evidenced by a decrease in p27 protein levels and cell death. The R-CLL subgroup expresses high ZAP-70 mRNA levels which portends a worse clinical course. In this subgroup, ZAP-70 kinase activity may provide an additional tonic signal that pushes CLL B-cells into late G0/early G1. At this stage, DPP2 protease activity is no longer required for survival. DPP2 inhibition may serve as an easy-to-perform prognostic test, as well as a novel therapeutic approach.

Author notes

Disclosure: No relevant conflicts of interest to declare.