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L. Patrussi et al.
cells, p66Shc expression increased in splenic leukemic cells from Eμ-TCL1 sick mice treated with 1 μM ibrutinib for 48 h, concomitant with increased STAT4 expression (Figure 1F,G) supporting the notion that the therapeutic effects of ibrutinib are associated with its STAT4/p66Shc- elevating activity.
p66Shc deficiency accelerates leukemogenesis in Eμ-TCL1 mice
Our results suggest that the p66Shc defect observed in leukemic CLL and Eμ-TCL1 cells may be implicated in dis- ease pathogenesis. To test this hypothesis, we transferred the p66shc-/- allele into Eμ-TCL1 mice (Online
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Figure 1. p66Shc expression decreases during leukemia progression in tumoral cells from Eμ-TCL1 mice and can be restored by ibrutinib treatment. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of p66Shc mRNA in B1a, B1b, B2 and total mature B lymphocytes purified from four wildtype (WT) mice and in leukemic cells purified from five Eμ-TCL1 sick mice. The relative gene transcript abundance was determined on triplicate samples using the DDCt method and normalized to GAPDH. (B, E). qRT-PCR analysis of p66Shc (B) and STAT4 (E) mRNA in B lymphocytes purified from five WT mice and in leukemic cells purified from Eμ-TCL1 mice with mild (~20% CD5+CD19+ cells in peripheral blood) (n=6) or overt leukemia (≥50% CD5+CD19+ cells and white cell count >10.7x106/mL in peripheral blood) (n=5). The relative gene transcript abundance was determined on triplicate samples using the DDCt method. (C) Immunoblot analysis with anti-Shc and anti-STAT4 antibodies of postnuclear supernatants of leukemic cells purified from either WT (n=3) or Eμ-TCL1 mice with mild (n=3) or overt leukemia (n=3). The stripped filters were reprobed with anti-actin antibodies. (D) Correlation between the percentages of CD5+CD19+ cells and the mRNA levels of p66Shc in peripheral blood samples obtained from Eμ-TCL1 mice at different disease stages (n=12). (F) qRT-PCR analysis of p66Shc (left) and STAT4 (right) mRNA in leukemic cells purified from spleens of Eμ-TCL1 sick mice (n=4) incubated for 48 h with either dimethylsulfoxide (DMSO) (absolute cell viability: 88.4 ± 3.2% of annexin V-/propidium iodide- cells) or 1 μM ibrutinib (absolute cell viability: 84.9 ± 2.9% of annexin V-/propidium iodide- cells). The relative gene transcript abundance was determined on triplicate samples using the DDCt method. (G) Immunoblot analysis with anti-Shc and anti-STAT4 antibodies of postnuclear supernatants of leukemic cells purified from spleens of Eμ-TCL1 sick mice (n=3) incubated for 48 h with either DMSO or 1 μM ibrutinib. The stripped filters were reprobed with anti- actin antibodies. Mean ± standard deviation. One-way analysis of variance (ANOVA), multiple comparisons. ****P≤0.0001; ***P≤0.001; **P≤0.01; *P≤0.05
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haematologica | 2019; 104(10)