Chronic lymphocytic leukemia (CLL) is a paradigmatic malignancy in which both cell-extrinsic (microenvironmental) and cell-intrinsic (genetic) factors contribute not only to the pathogenesis of the disease but also to disease evolution and outcome.21 In more recent years, the genomic landscape of CLL has been unraveled with the identification of “driver” gene mutations associated with clinical aggressiveness and chemorefractory disease, such as ATM, BIRC3, NOTCH1, NFK-BIE, SF3B1 and TP53.53 In addition to genetic aberrations, we also know that the B-cell receptor (BCR) immunoglobulin plays a pivotal role in driving the disease onset and evolution. The somatic hypermutation status of IGHV genes divides patients into two major clinical subgroups of CLL, with IGHV-unmutated patients displaying a more rapidly progressing disease and an overall poor survival compared with IGHV-mutated patients.76 The importance of BCR signaling was reinforced with the successful introduction of BCR inhibitors (e.g. ibrutinib) in the treatment of CLL, which abrogate downstream BCR signaling and are effective in patients with a poor prognosis, i.e. patients with TP53 aberrations and/or unmutated IGHV genes.8 Finally, the micromilieau strongly contributes to CLL cell survival through direct/indirect interactions with different cell types (e.g., T cells, stromal cells) and receptors (e.g. CRCX4 and CCR7), in particular within the proliferation centers in secondary lymphoid organs.9
One of the few existing in vivo CLL models is the transgenic Eμ-TCL1 mouse, which recapitulates clinically aggressive human CLL.10 Thanks to this model, it is possible to study the different phases of disease development. In the current issue of Haematologica, Petrussi et al. have taken advantage of the Eμ-TCL1 mouse model to investigate the potential role of p66Shc deficiency in CLL pathobiology.11 The authors previously reported a significant association between p66Shc deficiency and dismal outcome, potentially linking this event to clinical aggressiveness.12 p66Shc is a Shc family adaptor that promotes production of reactive oxygen species (ROS), which in turn activate cell apoptosis. p66Shc is also known to act as a negative regulator of BCR signaling and to regulate lymphocyte homing by controlling the expression of different chemokine receptors (e.g., CXCR4 and CCR7).13
In their present study, Patrussi and colleagues first showed that leukemic B cells from Eμ-TCL1 mice with more advanced disease displayed lower levels of p66Shc expression compared with the levels in normal B cells.11 STAT4, an essential transcription factor for p66Shc,14 was also downregulated in Eμ-TCL1 mice. Notably, by treating leukemic Eμ-TCL1 splenic cells with ibrutinib they could restore p66Shc expression along with STAT4 expression, similar to what they had reported earlier in primary CLL.13
Next, by crossing the Eμ-TCL1 mice with p66Shc deficient mice, to produce Eμ-TCL1/p66Shc mice, the latter demonstrated a markedly more rapid increase of CD5/CD19 cells in peripheral blood, a 2-month earlier disease onset and shorter overall survival compared to Eμ-TCL1 mice.11 When leukemic cells from Eμ-TCL1/p66Shc mice, which expressed higher Bcl2 levels, were treated with fludarabine, they were more resistant than Eμ-TCL1 cells, indicating that p66Shc loss also contributes to decreased chemo-sensitivity.
The Eμ-TCL1 model also offers the unique possibility to study the pattern of disease infiltration. In Eμ-TCL1/p66Shc mice, an increased accumulation of leukemic cells was observed in both nodal and extranodal sites; higher percentages and proliferation rates of leukemic cells were preferentially seen in lymph nodes, liver and lung, but not in spleen and bone marrow. While expression of CXCR4 was unchanged in leukemic cells, the expression of CCR7, a key lymph node B-cell homing receptor, CCR2 (associated with both lung and liver homing) and CXCR3 (linked to lung homing) was higher in Eμ-TCL1/p66Shc leukemic cells than in cells from Eμ-TCL1 mice.11 Hence, a more efficient accumulation in nodal and extranodal sites appears to depend on distorted chemokine receptor expression due to p66Shc deficiency.
p66Shc deficiency was also confirmed in primary CLL cells, in particular in IGHV-unmutated CLL; however, the authors could not find any correlation with 13q deletion or TP53 aberrations. Similar to Eμ-TCL1/p66Shc mice, both CCR2 and CXCR3 were overexpressed in IGHV-unmutated CLL, and p66Shc re-expression in CLL cells resulted in decreases in CCR2 and CXCR3 mRNA expression. The number and size of infiltrated lymph nodes and presence of spleen and/or liver enlargement were also higher in patients with low p66Shc mRNA levels. Interestingly, the authors observed that p66Shc expression was higher in CLL patients with a known response to second-line ibrutinib treatment than in patients who did not respond.
Finally, Patrussi et al. demonstrated a correlation between decreased ROS production and p66Shc deficiency both in CLL cells and Eμ-TCL1/p66Shc cells.11 Using transfection experiments, wildtype p66Shc-expressing transfectants, but not the ROS-defective mutant, showed lower CCR2 and CXCR3 expression compared to control cells, implying that p66Shc directly modulates CCR2 and CXCR3 expression through elevation of ROS.
Based on the novel results from this study, p66Shc deficiency in Eμ-TCL1 mice was shown to accelerate disease onset and progression to an aggressive phenotype. The authors also followed organ selectivity which correlated with deregulation of specific chemokine receptors. Importantly, p66Shc expression could be restored by ibrutinib treatment, along with enhanced chemo-sensitivity. Considering the potential role of p66Shc deficiency preceding a more aggressive phase of the disease, it will now be important to follow CLL patients longitudinally and in relation to therapy in order to determine if p66Shc could be used as a potential biomarker. Could p66Shc expression predict a shift from a more indolent to a more aggressive disease in need of therapy? Could p66Shc be monitored after treatment with chemo-immunotherapy or targeted therapy as an early indicator of disease progression/chemo-refractoriness? From a biological point of view, it will also be important to identify what cellular/molecular factors are involved in and lead to p66Shc downregulation. Do other cell types in the CLL microenvironment influence p66Shc levels? Is there any correlation with gene mutations affecting key cellular pathways and processes linked to an aggressive phenotype or chemo-resistance? While the study by Petrussi et al. using Eμ-TCL1/p66Shc mice has provided us with important insights, further studies are now necessary to investigate the potential clinical role of p66Shc deficiency, preferably in uniformly treated cohorts and involving novel therapies.
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