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Eμ-TCL112,13 and p66Shc-/- C57BL/6J (C57/p66-/-)14 mice were
crossed to generate Eμ-TCL1/p66Shc-/- mice: the screening strategy
is illustrated in Online Supplementary Figures S1 and S2. C57BL/6J
and C57/p66-/- mice were used as controls. Mice with ≥10%
peripheral blood CD5+CD19+ cells were considered to have devel-
oped leukemia. Overt leukemia was defined as reported else-
13 where.
Cell lines, patients and healthy donors
Transfectants generated using the CLL-derived B-cell line MEC115 and expressing human full-length p66Shc or the p66ShcQQ mutant were described previously.8 p66shc silencing in an Epstein-Barr virus (EBV) B-cell line was performed as described elsewhere.10 Peripheral blood samples were collected from 157 treatment-naïve CLL patients and five CLL patients subjected to pharmacological treatments (Online Supplementary Table S1). B cells from 15 buffy coats were used as healthy population controls. B cells were purified and transfected as described previously.8
Immunophenotyping of leukemic cells
Single-cell suspensions from mouse peripheral blood, peritoneal wash, spleen, bone marrow, lymph nodes, liver and lung were depleted of erythrocytes by hypotonic lysis and incubated with mouse Fc-Block for 15 min at 4°C. Murine leukemic cells and B cells purified from CLL patients and healthy donors were stained (antibodies and reagents listed in Online Supplementary Table S2) and subjected to flow cytometry (Guava Easy Cyte cytometer, Millipore).
RNA purification and polymerase chain reactions
Total RNA was extracted and retrotranscribed as described pre- viously.8 Real-time polymerase chain reactions (PCR) (primers list- ed in Online Supplementary Table S3) were performed using GoTaq Long PCR Master Mix (Promega) and results were processed and analyzed as described elsewhere.11
haematologica | 2019; 104(10)
p66shc deletion exacerbates leukemia in TCL1 mice
regulates B-cell survival by modulating the expression of several Bcl-2 family members and inhibiting the activation of the pro-survival kinase Akt.5,6 p66Shc also participates in B-cell trafficking by controlling CCR7, CXCR4 and S1PR1 surface expression both transcriptionally and post- translationally.7,8 Moreover, p66Shc attenuates CXCR4 and CXCR5 signaling.9 CLL cells have a defect in the expres- sion of p66Shc6 and its transcription factor STAT410 which is causal to their extended survival,6,7,11 suggesting a link between p66Shc deficiency and the pathogenesis of CLL.
Overexpression of TCL1 driven by the IgM heavy chain enhancer (Eμ-TCL1) in mice results in the development of a B-cell leukemia that recapitulates aggressive CLL.12 Here we have addressed the effect of p66Shc deficiency on the onset, development and outcome of CLL by generating a Eμ-TCL1/p66Shc-/- mouse. We showed that p66Shc dele- tion in Eμ-TCL1 mice resulted in accelerated leukemogen- esis and enhanced disease aggressiveness, with enhanced nodal and extranodal infiltration. Our data provide direct evidence that p66Shc deficiency concurs to CLL pathogen- esis and highlight p66Shc expression as a relevant disease target.
Methods
A detailed description of the methods is available in the Online Supplementary Data file.
Mice
Cell treatment, apoptosis and reactive oxygen species measurement, immunoblot and chemotaxis assays
Freshly isolated normal and leukemic mouse B cells were treat- edwith50μMH2O2 for24h,1μMibrutinibfor48h,or35μM fludarabine phosphate for 16 h. Dimethylsulfoxide was used as a control. Apoptosis was measured by flow cytometry on FITC- labeled annexin-V -stained CD5+IgM+ cells. Intracellular ROS were measured by flow cytometry in cells labeled for 30 min at 37°C with 5 μM CM-H2DCFDA.8
Immunoblots and chemotaxis assays (antibodies and chemokines listed in Online Supplementary Table S2) were carried out as reported previously.16
Histopathology and immunohistochemistry
Tissues, peripheral blood and peritoneal wash were collected and processed as detailed in the Online Supplementary Methods.
Statistical analyses
One-way analysis of variance (ANOVA) with a post-hoc Tukey test was used for experiments in which multiple groups were compared. Mann-Whitney rank-sum tests were performed to determine the significance of differences between two groups. Survival curves and medians were calculated within subgroups with the Kaplan-Meier method. A log-rank test was used to com- pare differences between estimated survival curves. Statistical analyses were performed using GraphPad Software (La Jolla, CA, USA). P values <0.05 were considered statistically significant.
Study approval
Experiments were approved by the Institutional Review Board and the local Ethics Committee.
Results
p66Shc expression decreases during leukemia progression in tumoral cells from Eμ-TCL1 mice
CLL cells have a profound reduction in p66Shc expres- sion, which is more severe in patients with an unfavorable prognosis.6 As Eμ-TCL1 mice are a model of aggressive CLL,17 we investigated whether the p66Shc defect in CLL cells is recapitulated in leukemic Eμ-TCL1 cells. p66Shc mRNA was quantified in splenic leukemic cells from Eμ- TCL1 mice with overt leukemia (≥50% peripheral blood CD5+CD19+ cells and a white blood cell count above the normal range; see Online Supplementary Methods).13 The analysis was extended to B1a cells from C57BL/6 mice, the normal CD5+ B-cell counterpart,18 and to the two other mature B-cell subsets, B1b and B2. While p66Shc was expressed at comparable levels in all normal B-cell subsets, tumoral B cells from mice with advanced disease expressed less p66Shc compared to normal B1a cells (Figure 1A). Interestingly, B1a cells from Eμ-TCL1 mice with milder disease (~20% peripheral blood CD5+CD19+ cells) had intermediate levels of p66Shc (Figure 1B,C), indicating that p66Shc expression declines during disease progression, as further supported by a >78% inverse correlation between p66Shc mRNA levels in CD5+CD19+ cells and the percent- age of leukemic cells in peripheral blood from the same mouse (Figure 1D). STAT4, a key transcription factor for p66Shc that is defective in CLL cells,10 was also downregu- lated in Eμ-TCL1 mice (Figure 1C,E).
The p66Shc expression defect in CLL cells can be res- cued by in vitro or in vivo treatment with the Btk inhibitor ibrutinib, used for CLL treatment.7,19 As for human CLL
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