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p66shc deletion exacerbates leukemia in TCL1 mice
Discussion
Here we used a genetic approach to specifically assess the outcome of p66Shc deficiency on CLL cell survival and disease onset and development. We showed that p66Shc deletion in Eμ-TCL1 mice results in accelerated leukemo- genesis and enhanced disease aggressiveness, with mas- sive nodal and extranodal infiltration. The enhanced abili- ty of leukemic p66Shc-/- cells to infiltrate organs was asso- ciated with increased expression of chemokine receptors that drive homing to the organs. p66Shc expression declined with disease progression in Eμ-TCL1 cells, simi- lar to human CLL. This defect could be restored by ibruti- nib treatment which enhanced the cells’ chemosensitivity. These results demonstrate in vivo that the p66Shc defect found in CLL cells concurs to CLL pathogenesis. Of note, p66Shc-/- mice spontaneously develop age-related autoim- munity,5 a feature frequently associated with CLL.1 Interestingly, p66Shc downregulation in CLL B cells induces the expression of the inhibitory molecule ILT3,39 suggesting that compensatory mechanisms might be oper- ational to restrain CLL cell responses.
The negative impact of p66Shc deletion on disease pro- gression and outcome in Eμ-TCL1 mice can be accounted for, at least in part, by the extended survival and chemore- sistance of leukemic cells, even when co-cultured with stromal cells as a surrogate pro-survival microenviron- ment. The p66Shc expression defect in CLL contributes to this biological behavior. p66Shc deficiency does indeed impinge on the Bcl-2 family balance in B cells, contributing to the shift of CLL cells towards survival, which correlates with chemoresistance and poor prognosis.40 The ROS-ele- vating activity of p66Shc4,14 underlies this latter’s ability to modulate the genes, several of which are redox-sensitive.41
The survival of CLL cells depends to a major extent on their ability to home to the pro-survival microenvironment of bone marrow and secondary lymphoid organs. This process is orchestrated by homing receptors responding to local chemokines and egress receptors responding to lymph and blood S1P.3 p66Shc is a central part of this circuitry which it affects by: (i) modulating CCR7 and S1PR1 expres- sion in opposite directions in a ROS-dependent fashion;8 (ii) modulating CCR7 and CXCR4 by slowing down their endosomal recycling;7 and (iii) attenuating CXCR4 and CXCR5 signaling by recruiting the phosphatases SHP-1 and SHIP-1 close to the activated receptors.9 The p66Shc defect in CLL cells has a major impact on these processes, resulting in enhanced responses to the chemokines of the lymphoid niche and impaired response to S1P.7,8 This imbalance is expected to contribute to the lymphadenopathy and chemoresistance observed in a significant proportion of CLL patients, and indeed the levels of CCR7 are significant- ly higher and those of S1PR1 lower in CLL patients with clinical lymphadenopathy.8 We showed that the levels of p66Shc in leukemic cells are inversely related to both the number and size of infiltrated lymph nodes in CLL patients. The results obtained in Eμ-TCL1/p66Shc-/- mice, showing
massive lymph node accumulation during disease progres- sion, provide experimental evidence that p66Shc deficiency promotes the nodal leukemic cell accumulation in CLL.
p66Shc deficiency also results in a striking extranodal accumulation of leukemic cells, with a preference for liver and lung, the most frequent extranodal target sites in CLL.21,22 The ROS-related ability of p66Shc to modulate the expression and function of CCR2 and CXCR3, which drive neoplastic B-cell homing to liver and lung where the respective ligands are expressed,42,43 may account for the enhanced ability of leukemic Eμ-TCL1/p66Shc-/- cells to colonize these organs. Interestingly, CCR2 and CXCR3 are overexpressed in CLL cells (as shown in this study and reported by Trentin et al.44 for CXCR3). We show that p66Shc reconstitution in CLL cells reverts these abnormal- ities, validating in human CLL our finding that p66Shc deficiency contributes to CCR2 and CXCR3 overexpres- sion in leukemic Eμ-TCL1/p66Shc-/- cells.
p66Shc expression declines during disease progression in Eμ-TCL1 mice, until its almost complete loss in mice with overt leukemia, paralleling the progressive decrease in fludarabine sensitivity of tumoral cells documented pre- viously.20 p66shc transcription is largely controlled in sever- al primary and transformed cells, including T cells, by his- tone deacetylation and cytosine methylation in a CpG island within the promoter.45,46 Although methylation increases in Eμ-TCL1 mice during disease development,47 it is unlikely that methylation of the p66shc promoter caused its progressive silencing, as p66Shc expression is not epigenetically silenced in B cells.6 Rather, in these cells p66shc is transcriptionally regulated by STAT4, which is defective in CLL cells.10 Interestingly, p66Shc can be restored both in CLL cells7 and in leukemic Eμ-TCL1 cells (Figure 1F,G) by treatment with ibrutinib, which also pro- motes STAT4 expression in leukemic Eμ-TCL1 cells (Figure 1F,G). Ibrutinib modulates the expression of genes downstream of Btk in the BCR and CXCR4 pathways,48,49 which are implicated in CLL, suggesting that STAT4 and its target p66Shc may be regulated through these path- ways. While this remains to be established, considering the pleiotropic role of p66Shc in B-cell survival and traf- ficking our finding suggests that direct or indirect STAT4 agonists that enhance the activity of residual STAT4 in CLL cells may normalize p66Shc expression and over- come chemoresistance in CLL. Our finding that inter- leukin-12, which activates STAT4, restores p66Shc expres- sion in CLL cells10 supports this hypothesis. Collectively, our findings underscore the pathological outcome of p66Shc deficiency in CLL and highlight the chemokine receptor network as a central target of its activity.
References
1. Dighiero G, Hamblin TJ. Chronic lympho- cytic leukaemia. Lancet. 2008;371(9617): 1017–1029.
2. Ortiz-Maldonado V, Mozas P, Delgado J.
The biology behind B-cell lymphoma 2 as a target in chronic lymphocytic leukemia. Ther Adv Hematol. 2016;7(6):321–329.
3. Burger JA. Nurture versus nature: the microenvironment in chronic lymphocytic leukemia. Hematol Am Soc Hematol Educ
Progr. 2011;2011(1):96–103.
4. Giorgio M, Migliaccio E, Orsini F, et al.
Electron transfer between cytochrome c and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis. Cell. 2005;122(2):221–233.
Acknowledgments
The authors thank Carlo M. Croce for providing Eμ-TCL1 mice and Sonia Grassini for technical assistance. This work was supported by grants from AIRC (IG-20148) and ITT-Regione Toscana to CTB, AIRC (IG-19236) to DGE, AIRC (IG-15286) to GS and AIRC (IG-15397) to LT.
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