A Pyrazolo[3,4-d]pyrimidine compound inhibits Fyn phosphorylation and induces apoptosis in natural killer cell leukemia

Abstract

Natural killer (NK) cell neoplasms are characterized by clonal proliferation of cytotoxic NK cells. Since there is no standard treatment to date, new therapeutic options are needed, especially for NK aggressive tumors. Fyn tyrosine kinase has a key role in different biological processes, such as cell growth and differentiation, being also involved in the pathogenesis of hematologic malignancies. Our previous studies led us to identify 4c pyrazolo[3,4-d]pyrimidine compound capable of inhibiting Fyn activation and inducing apoptosis in different cancer cell lines. Here we investigated the presence of Fyn and the effect of its inhibitor in NK malignant cells. Firstly, we showed Fyn over-expression in NK leukemic cells compared to peripheral blood mononuclear cells from healthy donors. Subsequently, we demonstrated that 4c treatment reduced cell viability, induced caspase 3-mediate apoptosis and cell cycle arrest in NK cells. Moreover, by inhibiting Fyn phosphorylation, 4c compound reduced Akt and P70 S6 kinase activation and changed the expression of genes involved in cell death and survival in NK cells. Our study demonstrated that Fyn is involved in the pathogenesis of NK leukemia and that it could represent a potential target for this neoplasm. Moreover, we proved that Fyn inhibitor pyrazolo[3,4-d]pyrimidine compound, could be a started point to develop new therapeutic agents.

Keywords: Fyn tyrosine kinase; NK cells; kinase inhibitor; natural killer large granular lymphocyte leukemia.

Figure 1. Fyn is over-expressed in NK malignant cells

(A) qRT-PCR of Fyn mRNA in PBMCs from 10 HDs and from 8 patients with CLPD (UPN: 1–8). Statistical significance was determined using an unpaired t test and P-value is indicated by asterisk: ***p < 0.001. (B) WB analysis of Fyn in 3 HDs, 3 CLPD patients (UPN: 6–7) and two NK cell lines, KHYG1 and NK92. Quantification of Fyn protein levels was normalized with β-actin bands.

Figure 2. 4c compound reduced cell viability inducing apoptosis and cell cycle arrest in NK leukemic cells

(A) Viability of KHYG1, NK92, 3 PBMCs from HDs (HDs) and NK primary cells sorted from 3 HDs (HD-NK cells) was evaluated by MTS assay after treatment with 4c compound at different concentration (2–10 μM) for 24–72 hours. Results are expressed as percent of cell viability normalized to DMSO-treated control cells. The bar-graphs represent mean with S.D. from three independent experiments. (B) Apoptosis and (C) cell cycle analysis were evaluated by flow cytometer in NK cell line after 4c or DMSO vehicle treatment at 4 μM for 24 hours. Dot plots and cell cycle histograms show a single representative experiment, the bar-graphs represent mean with S.D. from three independent experiments. P-value is indicated by asterisk: ***p < 0.001.

Figure 3. Inhibition on Fyn phosphorylation by 4c compound decreased Akt and P70 phosphorylation

WB analysis of (A) phospho-Fyn and (B) total and phosphorylated Akt and P70 S6 kinase in NK cell line treated with 4c compound compared to control. Protein levels were normalized on β-actin signals. Bar-graphs represent mean with S.D. from three independent experiments. P-values are indicated by asterisk: *p < 0.05, **p < 0.01.

Figure 4. Activation of apoptotic pathways in 4c treated cell line

(A–B) Bar-graphs of the first cluster up-regulated (A) and down-regulated (B) genes resulting by DAVID bioinformatic tool in GEP analysis of KHYG1 treated with 4c compound or with DMSO control. (C) qRT-PCR of Survivin, CFLAR and DFFA in KHYG1 treated vs control. Bar-graphs represent mean with S.D. from three independent experiments. (D) Bar-graphs of protein levels resulted from apoptotic protein array and (E) WB analysis of caspase 3 in KHYG1 treated vs control. P-values are indicated by asterisk: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. 4c treatment reduced viability and induced caspase 3-mediated apoptosis in PBMC from CLPD patients

(A) Trypan blue count was performed in primary cells, isolated from 3 HDs and 3 CLPD patients (UNP: 6–8) and (B) Caspase 3/7 activity assay on 3 CLPD patients (UNP: 6–8) after 24 hours of treatment with 4c compound at 4 μM concentration. P-values are indicated by asterisk: *p < 0.05, **p < 0.01.

Figure 6. 4c compound treatment reduced cell viability and proliferation, induced caspase 3-mediated apoptosis and cell cycle arrest in primary NK leukemic cells

On 4c treated or not primary NK cells sorted from PBMCs of 3 NK-CLPD patients (UPN: 6–8) (A) Trypan blue count, (B) PKH67 proliferation test, (C) Apoptosis and (D) Caspase 3/7 activity assay, (E) Cell cycle analysis were performed. Proliferation, apoptosis and caspase 3/7 level were analyzed on CD56⁺/CD16⁺ and CD56⁺/CD16⁻ NK cell populations. P-values are indicated by asterisk: *p < 0.05, **p < 0.01. (MFI= mean fluorescent intensity).

Figure 7. 4c compound treatment induced phenotype changes in NK primary cells

On 4c primary NK cells sorted from PBMCs of 3 CLPD patients (UPN: 6–8) (A) CD38 and (B) CD25 expression were evaluated after 4c treatment. P-values is indicated by asterisk: ***p < 0.001. (MFI = mean fluorescent intensity)