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p66shc deletion exacerbates leukemia in TCL1 mice
Supplementary Figure S1A,B).12 As expected, Eμ- TCL1/p66Shc-/- B cells did not express p66Shc (Online Supplementary Figure S1C,D) while expressing wildtype p53, similar to Eμ-TCL1 cells (Online Supplementary Methods and Online Supplementary Table S3).20 Eμ- TCL1/p66Shc-/- mice developed a CLL-like disease similar to that occurring in Eμ-TCL1 mice. A large lymphocyte population, associated with the characteristic Gumprecht shadows, was evident in peripheral blood smears from sick mice (Online Supplementary Figure S1E). Flow cytomet- ric analysis of peripheral blood samples from 6- and 9- month old mice revealed a discrete population of CD5+ B cells, which is the hallmark of disease development in Eμ- TCL1 mice (Online Supplementary Figures S1F and S2).12 Similar to Eμ-TCL1 mice, leukemic Eμ-TCL1/p66Shc-/- mice eventually became visibly ill, showing labored breathing and splenomegaly (Online Supplementary Figure S1G and Online Supplementary Table S4) that necessitated euthanasia.
To address the impact of p66Shc deficiency on disease onset and progression, we followed leukemia develop- ment in 87 Eμ-TCL1 and 134 Eμ-TCL1/p66Shc-/- mice by immunophenotyping monthly peripheral blood samples. Eμ-TCL1/p66Shc-/- mice showed higher white blood cell counts and higher CD5+CD19+ cell percentages in periph- eral blood compared to those in Eμ-TCL1 mice (Figure 2A,B; Online Supplementary Figure S1G). Moreover disease progression, assessed as rate of increase in the percentage of CD5+CD19+ cells, was faster in Eμ-TCL1/p66Shc-/- mice (Figure 2C).
Disease incidence, defined as the percentage of sick mice (>10% peripheral blood CD5+CD19+ cells vs. ~5% in healthy C57BL/J6 mice) (Online Supplementary Figure S3) at defined time points, was significantly higher in Eμ- TCL1/p66Shc-/- mice than in Eμ-TCL1 mice (Figure 2D). Moreover, p66Shc deficiency led to an earlier onset of dis- ease, which was detected ~2 months earlier in Eμ- TCL1/p66Shc-/- mice (Figure 2D) and resulted in a shorter lifespan (Figure 2E). Hence p66Shc deficiency in Eμ-TCL1 mice accelerates disease onset and development as well as its progression to an aggressive presentation.
p66Shc deficiency enhances leukemic cell chemoresistance
Consistent with the ability of p66Shc to modulate the expression of pro- and anti-apoptotic Bcl-2 family mem- bers,6 leukemic Eμ-TCL1/p66Shc-/- cells expressed higher and lower levels of Bcl-2 and Bax, respectively, compared to levels in their Eμ-TCL1 counterparts (Figure 3A,B). At variance, Mcl-1 expression was comparable (Online Supplementary Figure S4). Accordingly, leukemic cells from sick Eμ-TCL1/p66Shc-/- mice were more resistant to flu- darabine treatment, also when co-cultured with OP9 stro- mal cells to simulate the pro-survival stromal microenvi- ronment (Figure 3C). Hence p66Shc deficiency in Eμ- TCL1 leukemic cells promotes these cells’ survival, which likely contributes to the faster disease development and unfavorable outcome in Eμ-TCL1/p66Shc-/- mice. This is supported by the higher sensitivity to fludarabine of leukemic cells from Eμ-TCL1 mice with milder disease (<35% peripheral blood CD5+CD19+ cells) compared to leukemic cells from mice at later disease stages (≥35% peripheral blood CD5+CD19+ cells) (Figure 3D), which may be accounted for, at least in part, by their higher residual p66Shc expression (Figure 1B,C). Consistent with
this notion, tumoral Eμ-TCL1/p66Shc-/- cells were less sen- sitive to fludarabine treatment, independently of disease stage (Figure 3D). Hence the reduction of p66Shc expres- sion in Eμ-TCL1 mice during disease development con- tributes to the decreased chemosensitivity of leukemic cells.
p66Shc deficiency in Eμ-TCL1 mice is associated
with nodal and extranodal leukemic cell accumulation
Leukemic cells accumulate in the peritoneal cavity of Eμ- TCL1 mice at early stages of disease, subsequently coloniz- ing spleen, bone marrow and lymph nodes as well as extra- nodal sites.12 p66Shc modulates the surface levels of recep- tors that regulate homing to and egress from secondary lymphoid organs,8,7 suggesting that its deficiency might con- tribute to leukemic cell accumulation in secondary lym- phoid organs and infiltration into non-lymphoid organs. The extent of organ infiltration by leukemic cells was meas- ured in spleen, bone marrow and lymph nodes from Eμ- TCL1 and Eμ-TCL1/p66Shc-/- mice with ~60% leukemic cells in peripheral blood. The analysis was extended to liver and lung, as infiltration in these organs has been document- ed in CLL,21,22 as well as to the peritoneal infiltrate.
Flow cytometric analysis of CD5+CD19+ cells revealed higher percentages of leukemic cells in lymph nodes of Eμ-TCL1/p66Shc-/- mice than in those of Eμ-TCL1 mice (Figure 4A). This was confirmed by hematoxylin & eosin staining and immunohistochemical analysis of the same organs, which highlighted massive lymph node infiltra- tion of Eμ-TCL1/p66Shc-/- mice by small lymphoid-like B220+ cells (Figure 4A). At variance, leukemic cell accumu- lation in spleen and bone marrow was comparable (Online Supplementary Figure S5 and Online Supplementary Table S4), suggesting that Eμ-TCL1/p66Shc-/- tumoral cells show organ selectivity.
Leukemic cell infiltrates in the liver and lung were found to be more substantial in Eμ-TCL1/p66Shc-/- mice than in Eμ-TCL1 mice and were frequently associated with loss of organ architecture (Figure 4B,C). Increased peritoneal leukemic cell accumulation was also observed in Eμ- TCL1/p66Shc-/- mice (Figure 4D). Interestingly, flow cyto- metric analysis of the proliferation marker Ki-67 revealed a higher proliferation rate of leukemic Eμ-TCL1/p66Shc-/- cells in lymph nodes, liver and lung compared to their Eμ- TCL1 counterparts (Online Supplementary Figure S6), con- sistent with the anti-mitogenic function of p66Shc.23
by Eμ-TCL1/p66Shc
expression of homing receptors
Increased colonization of nodal and extranodal sites
-/-
cells is associated with higher
The chemokine receptors CXCR4 and CCR7 contribute to the pathogenesis of CLL by modulating leukemic B-cell homing to secondary lymphoid organs and bone mar- row.3,24,25 p66Shc modulates surface expression of homing and egress receptors both transcriptionally and post-trans- lationally in normal and CLL B cells,7,8 suggesting that the increased colonizing ability of leukemic Eμ-TCL1/p66Shc-/- cells may be caused by imbalanced expression of these receptors. Splenic leukemic cells from Eμ-TCL1 and Eμ- TCL1/p66Shc-/- mice with overt leukemia were analyzed by quantitative real-time PCR and flow cytometry for expression of chemokine receptors that regulate CLL cell homing to and residency in lymphoid organs, namely CXCR4, CCR7 and S1PR1. Splenic B cells from C57BL6/J and C57BL6/J/p66Shc-/- mice were used as controls.
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