ZAP-70 Disrupts Dipeptidyl Peptidase 2 (DPP2)-Regulated Quiescence in Chronic Lymphocytic Leukemia (CLL).

Abstract 1251

Poster Board I-273

CLL is a common hematologic malignancy with heterogeneous outcomes. Overexpression of ZAP-70 and unmutated B-cell receptor (BCR) heavy chain gene (IgV_H) confer an adverse prognosis. While it is accepted that ZAP-70 augments BCR signaling in CLL B-cells, it remains unclear how ZAP-70 contributes to disease propagation. Peripheral blood CLL B-cells accumulate in G₀. We have previously shown that CLL B-cells express DPP2, a serine protease involved in maintenance of quiescence of resting but not activated lymphocytes. We identified two subsets of CLL: sensitive CLL (S-CLL), where CLL B-cells undergo apoptosis upon inhibition of DPP2, and resistant CLL (R-CLL), where inhibition of DPP2 does not cause cell death. Here we sought to validate our preliminary observation and establish the role of ZAP-70 and BCR signaling in resistance to apoptosis in CLL.

The patient cohort included 152 subjects with B-CLL from the Hematology clinics at Tufts Medical Center, Dana-Farber Cancer Institute (both in Boston, MA), and the Lahey Clinic (Burlington, MA). IgV_H mutational status, ZAP-70 expression and history of treatment were analyzed. CLL B-cells were isolated from peripheral blood with standard Ficoll-Hypaque technique. Cells were treated with ValboroPro (VbP, Point Therapeutics), a non-specific inhibitor of DPPs, or AX8819 (ActivX), a DPP2-specific inhibitor. To interfere with ZAP-70, cells were treated with 17-Allylaminodemethoxygeldanamycin (17-AAG, Calbiochem), an inhibitor of hsp90, a protein involved in stabilization of ZAP-70. For apoptosis analysis cells were stained with propidium iodide and Annexin V at 16 h of incubation and assayed by flow cytometry. A CBA assay was used to measure tyrosine-phosphorylated p72^Syk and ZAP-70. Expression of p27 and p130 proteins was assessed by western blot analysis.

Of 152 CLL patients 99 were males (65.1%). Median age was 63 years. Median follow up was 6 years. When the study samples were obtained, 107 patients (70.4%) were untreated. In apoptosis assays, 97 (63.8%) samples were categorized as S-CLL and 55 (36.2%) as R-CLL. The small molecule inhibitor data correlated with DPP2 anti-sense experiments. Patients with R-CLL were more likely to receive treatment of their disease and had a shorter treatment-free interval from disease diagnosis compared with S-CLL (HR=4.79, 95% CI, 4.7 to 15.2; p<0.0001).

In the R-CLL subgroup, B-CLL cells also exhibited unmutated IgV_H and expressed high level of ZAP-70 (p<0.001). R-CLL B-cells exhibited higher level of p72^Syk phosphorylation compared with S-CLL (p<0.05), suggesting that those cells are partially activated. Meanwhile, S-CLL B-cells had high p27 and p130 protein level, characteristic of a quiescent state. ZAP-70 was phosphorylated at a similar level between the two subsets, consistent with earlier observations that ZAP-70 does not depend on its phosphorylation to enhance BCR signaling. Upon inhibition of DPP2, S-CLL B-cells became activated as evidenced by dramatic phosphorylation of p72^Syk. Concomitantly, p27 and p130 protein levels decreased indicating inappropriate cell cycle entry. Co-inhibition of DPP2 and hsp90 in R-CLL B-cells increased apoptosis by a mean of 8.1%, indicating that their survival is dependent on ZAP-70.

CLL can be categorized into two prognostic groups based on the susceptibility of B-cells to DPP2 inhibition-induced apoptosis. Resistance to apoptosis correlates with unmutated IgV_H and high ZAP-70 and is associated with an unfavorable disease course. The distinction between the two subsets of CLL stems from the aberration in the quiescence program. While S-CLL B-cells rest in true G_0, R-CLL B-cells are partially activated due to ZAP-70 co-stimulatory signal and escape apoptosis. Destabilization of ZAP-70 reverses the resistant phenotype. DPP2 inhibition alone or with concomitant inhibition of ZAP-70 warrants investigation as a therapeutic modality in CLL.