New SMAC-Mimetic Compounds Strongly Induce In Vitro Apoptosis of Human Lymphocytes From Patients with Chronic Lymphocytic Leukemia
Authors: Onida Francesco , Scavullo Cinzia , Servida Federica , Saporiti Giorgia , Seneci Pierfausto , Reda Gianluigi , Barcellini Wilma , Cortelezzi Agostino , Delia Domenico , Deliliers Giorgio Lambertenghi 
Published: October 2019

Abstract
Abstract 1835
Defective apoptosis has a central role both in development and progression of chronic lymphocytic leukemia (CLL). Indeed in this hematological malignancy the expression of inhibitor of apoptosis proteins (IAPs) has been shown to be significantly up-regulated. The most potent among IAPs is probably the X-linked IAP (XIAP), which is characterized by three highly conserved tandem BIR domains (BIR1-3) able to block various members of the family of cysteine proteases (caspases) known as the main effectors of the apoptotic process. BIR3 domain selectively targets caspase 9, an initiator caspase, whereas the linker region between BIR1 and BIR2 binds with executioner caspases 3 and 7, thus inhibiting the activity of both initiator and effector of apoptosis. Smac-DIABLO (Second Mitochondria-derived Activator of Caspases - Direct IAp Binding protein with LOw pI) is a protein released from the mitochondria antagonizing the activity of XIAP. Therefore, small molecules mimicking Smac (Smac-mimetics) can induce apoptosis in tumor cells by displacing the interaction between XIAP and caspases.
At the University of Milan, Center for biomolecular Interdisciplinary Studies and Industrial applications (CISI), new cell permeable Smac-mimetic compounds (monomers and dimers) binding to XIAP with high affinity were recently synthetized. Here we report the activity of a selection of these new agents in human lymphocytes from 32 CLL patients and from 6 healthy controls. The patient population included only subjects with active disease (11 females and 21 males; median age 72 years; Rai-Binet stage 0-A to II-B), who, at the time of samples collection, were either untreated or out of therapy since several months. Patients were characterized for the mutational IGVH status, cytogenetics (by FISH analysis), and the expression of both CD38 and ZAP 70. Following isolation from peripheral blood samples by density gradient centrifugation, mononuclear cells were treated for 16 hours at 37°C and 5% CO2 with low micromolar concentrations (1-10 microM) of a selection of our new Smac mimetic compounds, including 4 monomers (34, 55, 66 and 73), and 2 dimers (83, 85) which were have been previously shown to be active in leukemic cell lines overexpressing XIAP. Combination treatments with the proteasome inhibitor bortezomib (10nM) and with Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) (50 ng/ml) were also tested. Apoptosis rate was evaluated by flow cytometric analysis following annexin V and propidium iodide staining.
Overall, the monomers were more cytotoxic than the dimeric compounds on leukemic cells. The most active agent was the monomer Smac66, which, after 16 hours of treatment at a 10 microM concentration promoted apoptosis in up to 72,5% (median value 40% ranging from 11% to 72,5%) of CLL lymphocytes. In comparison to normal lymphocytes from healthy controls, where the median rate of apoptosis was 10.1% (range1.2% to 18%), the cytotoxic effect of the Smac66 compound was significantly higher (p=0.0004) in CLL cells. No synergistic effect was observed in combined treatment with bortezomib nor with TRAIL. Rate of apoptosis did not correlate with clinical parameters, cytogenetics, mutational IGVH status, and expression of CD38 and ZAP70 was observed, but the limited size of our patient population should be taken into account.
In conclusion, among our Smac mimetics selected molecules seem be very active in CLL lymphocytes, suggesting a promising therapeutic potential in this malignancy. Further molecular investigations as well as in vivo studies in animal models are currently ongoing respectively to better elucidate the mechanism of action of these compounds and to promote their preclinical development.

Disclosures:
No relevant conflicts of interest to declare.