A Pyrazolo[3,4-d]Pyrimidine Compound Reduces Fyn Phosphorylation and Induces Apoptosis in Large Granular Lymphocyte Leukemia Cells
Authors: Laurenzana Ilaria , Caivano Antonella , La Rocca Francesco , Trino Stefania , De Luca Luciana , D'Alessio Francesca , Tintori Cristina , Simeon Vittorio , Traficante Antonio , Teramo Antonella , Zambello Renato , Semenzato Gianpietro , Schenone Silvia , Botta Maurizio , Musto Pellegrino , Del Vecchio Luigi 
Published: October 2019

Abstract
Introduction
According to WHO classification, large granular lymphocyte leukemia (LGL leukemia) includes three entities: T-cell LGL leukemia, chronic NK lymphoproliferative disorders (NK-CLPD) and aggressive NK cell leukemia. Since no standard therapies for immature NK cell neoplasms have been established so far and the overall outcomes are dismal, new therapeutic options are needed.
A pyrazolo[3,4-d]pyrimidine library of compounds have already been studied in a previous work by Tintori et al. It has been demonstrated that one of these compounds proved to be efficient against some member of Src-family of nonreceptor protein-tyrosine kinases, in particular against Fyn. Its abnormal activity has been shown to be related to various hematological malignancies.
In this context we verified the effect of the pyrazolo[3,4-d]pyrimidine 4c compound on LGL leukemia cells and we investigated its mechanism of action.
Methods
The pyrazolo[3,4-d]pyrimidine 4c compound was tested in a time course MTS test (24, 48, 72 h) at different concentrations (0.1, 0.5, 1, 2, 4, 8, 10 µM) on NK leukemia cells (KHYG1 and NK92), hepatosplenic gamma-delta T cell lymphoma cells (Derl-2 and Derl-7), acute T cell leukemia cells (Jurkat), chronic myeloid leukemia cells (Jurl-MK1), acute myeloid leukemia cells (OCI), multiple myeloma cells (SKM-M1), cervical cancer cells (HeLa) and healthy stem cells (CD34+).
Fyn mRNA and protein expression was evaluated in all cell lines by quantitative RT-PCR and western blotting (WB). Fyn mRNA levels were evaluated in cells from 10 healthy controls, 10 T-LGL leukemia, 5 GD-LGL leukemia and 8 NK-CLPD patients by qRT-PCR.
Apoptosis and cell cycle studies were performed on KHYG1 after treatment with this compound at 4 µM for 24 h using cytometric analysis by labeling cells with annexin V and propidium iodide. Apoptosis pathway was investigated using proteome profiler human apoptosis array kit; pro-caspase 3, activated caspase 3, Akt and p70S6 kinase were validated by WB. Fyn immunoprecipitation was conducted on treated KHYG1 at 4 µM for 24 h.
Results
The pyrazolo[3,4-d]pyrimidine 4c compound reduced cell viability in KHYG1 cells (50% of reduction after 24 h of treatment at 4 µM), in Derl-2 cells (about 50% after 72 h at 4 µM), in Derl-7 (about 40% after 72 h at 8 µM), in Jurkat cells (50% after 72 h at 2 µM), in NK92 cells (40% after 72 h at 4 µM). No differences in cell viability were observed in Jurl-MK1, OCI, SKM-M1, HeLa and CD34+. To evaluate Fyn expression level, we performed qRT-PCR and WB showing that both transcript and protein levels were highest in NK and T cells (KHYG1>NK92>Jurkat>Derl-7>Derl-2) compared to other cells. Furthermore, we investigated the viability reduction in treated KHYG1 cells demonstrating that pyrazolo[3,4-d]pyrimidine 4c compound induced 45% of apoptosis caspase-3 mediated and cell cycle arrest in G2/M phase. In these cells we detected a reduction of Fyn phosphorylation after treatment and we also observed a decrease of total levels of Akt and p70S6 kinase, other two protein involved in apoptotic process and cell proliferation.
Interestingly, qRT-PCR data showed a significant over-expression of Fyn mRNA level also in T-LGL, GD-LGL and NK-CLPD patients compared to healthy controls (p<0.001).
Conclusion
In this study we demonstrated a higher Fyn expression in LGL leukemia patients compared to healthy controls. We also demonstrated an apoptotic effect of pyrazolo[3,4-d]pyrimidine 4c compound on NK leukemia cells and we provided in vitro preliminary evidences that Fyn is its possible cellular target.

Disclosures
Musto: Novartis: Honoraria; Celgene: Honoraria; Sanofi: Consultancy; Genzyme: Consultancy; Sandoz: Consultancy; Janssen: Honoraria; Mundipharma: Honoraria; Roche: Honoraria.